Vaccine to pathogenic immune activation cells during infections

ABSTRACT

A method for preventing an infectious disease in a subject in need thereof. In particular the method includes the administration of a combination, pharmaceutical combination, medicament or kit-of-parts including a first part including a CD8 vaccine specific for at least one infectious disease-related antigen, optionally a second part including an interferon alpha blocking agent, and a third part including a type III interferon and/or an agent stimulating the production of type III interferon.

Interestingly, the EC status suggests a low activation profile of Tcells and the existence of a unique MHC-1b/E-restricted CD8⁺ T cellpopulation able to suppress the early activation of pathogenic HIVantigen-presenting CD4⁺ T cells (Lu et al. (2016) Front. Immunol.7:134). Furthermore, recent advances in the field of SIV vaccinologyalso have highlighted the role of MHC-1b/E-restricted CD8⁺ T cellresponses in controlling SIV infection in rhesus macaques (Hansen et al.(2013) Science 24; 340(6135):1237874; Hansen et al. (2016) Science;351(6274), 714-20; Lu et al. (2012) Cell Rep. 2(6), 1736-46; Andrieu etal. (2014) Front Immunol. 5:297). These observations have suggestedalternative strategies for developing an HIV vaccine.

Indeed, since an activated state of the CD4⁺ T cell is a prerequisitefor productive HIV infection also in vivo, and thus replication inquiescent CD4⁺ T cells is essentially nonproductive and generallyabortive, it has been hypothesized that it might be possible to suppressviral replication by interfering with the CD4⁺ T cell activation.Therefore, several groups have tempted to suppress virus-specific CD4⁺ Tcell activation with vaccines that induce MHC-1b/E-restricted CD8⁺cells.

For example, Andrieu et al. have developed a vaccine able to induceMHC-1b/E-restricted CD8⁺ T cells in macaques. This vaccine consisted ofinactivated simian immunodeficiency virus (SIV) particles associatedwith a tolerogenic adjuvant, such as, for example, Lactobacillusplantarum. Although this vaccine strategy effectively immunized andinduced suppressive MHC-1b/E-restricted CD8⁺ T cells in Chinesemacaques, macaques of Indian origin that were immunized with the sameadjuvanted vaccine were not protected.

Hansen et al., by modifying cytomegalovirus (CMV) vectors determinantsthat control unconventional T cell priming, have shown that it waspossible to uniquely tailor the CD8⁺ T cell response in order tomaximize prophylactic or therapeutic protection. Specifically, it wasfound that the use of such rhesus cytomegalovirus vectors expressing SIVprotein in rhesus macaques (RMs) induces post-challenge sterileprotection against SIV. However, this protection was effective in only50% of vaccinated RMs.

Globally, these results have expanded the current paradigm from onefocused on a preventive HIV vaccine to one in which an immunotherapy forHIV/AIDS can be an essential part of the fight against this pandemic.Thus, in addition to a preventive vaccine, there remains a need for aneffective therapy to treat individuals living with HIV-1.

Based on the discovery of new biological properties of type IIIinterferon (IFN-III), the inventors propose to improve existing vaccine(i.e., induction of a suppressive MHC-1b/E-restricted CD8⁺ T cellpopulation) strategies for preventing or treating HIV and othersinfectious diseases.

In particular, the Applicant demonstrates that type III interferon maygreatly potentiate existing CD8 suppressive vaccines. Indeed, on thecontrary of type I interferons, type III interferons appear to have amore specialized role in innate antiviral defense and do not inhibit theinitiation of the adaptative immune reaction after HIV infection. Inparticular, IFN-III can prevent or limit virus replication withoutinhibiting the proliferation of CD4⁺ T cells that respond to HIVinfection.

The Applicant further demonstrates that the blockade of interferon alpha(IFN-α) as a supplement to a CD8 suppressive vaccine might potentiatetype III interferon and thus greatly improve the vaccine efficacy.Indeed, IFN-α is a paradoxical type I interferon cytokine that canprevent or limit virus replication while promoting a deleterious chronicimmune activation. Because chronic immune activation is necessary forHIV replication, IFN-α thus can play a deleterious role during HIVinfection. Moreover, as demonstrated by the inventors, IFN-α has ananti-proliferative activity that inhibits the proliferation of CD4⁺ Tcells, and therefore leads to immune decline, virus replication and AIDSin infected patients. Thus, by the blockade of IFN-α, the Applicant aimsto potentiate type III interferon activities, therefore promoting alower chronic immune activation and a better response of CD4⁺ T cells.

In the present invention, the Applicant thus provides a novel method forpreventing or treating an infectious disease in a subject in needthereof comprising administering to the subject:

-   -   1) a CD8 vaccine specific for at least one infectious        disease-related antigen,    -   2) optionally an interferon alpha blocking agent, and    -   3) a type III interferon and/or an agent stimulating the        production of type III interferon.

SUMMARY

The present invention relates to a method for preventing or treatingacquired immune deficiency syndrome (AIDS) in a subject in need thereof,comprising administering to the subject:

-   -   1) a CD8 vaccine specific for at least one human        immunodeficiency virus (HIV) antigen,    -   2) optionally interferon-alpha blocking agent, and    -   3) a type III interferon and/or an agent stimulating the        production of type III interferon.

In one embodiment, the type III interferon comprises at least one IFN-λselected from the group of IFN-λ1, IFN-λ2 IFN-λ3 and IFN-λ4, and whereinthe agent stimulating the production of type III interferon comprises atleast one TLR ligand, RIG-I ligand, and/or MDA5 ligand.

In one embodiment, the interferon-alpha blocking agent is selected fromthe group of: an agent neutralizing circulating alpha interferon, anagent blocking interferon-alpha signaling, an agent depleting IFN-αproducing cells, and/or an agent blocking IFN-α production, wherein theagent neutralizing circulating alpha interferon is selected from thegroup comprising active anti-IFN-α vaccine including antiferon orpassive anti-IFN-α vaccine including anti-IFN-α antibodies or anti-IFN-αhyper-immune serum, wherein the blocking agent of interferon-alphasignaling is selected from the group of anti-type I interferon R1 or R2antibodies or from interferon-alpha endogenous regulators includingSOSC1 or aryl hydrocarbon receptors, wherein the agent depleting IFN-αproducing cells is an agent depleting plasmacytoid dendritic cells(pDCs), and wherein the agent blocking IFN-α production is an agentblocking the production of IFN-α by pDCs.

In one embodiment, the CD8 vaccine elicits or comprises suppressorMHC-1b/E-restricted CD8⁺ T cells.

In one embodiment, the CD8 vaccine elicits or comprises suppressorMHC-1b/E-restricted CD8⁺ T cells, and wherein the suppressorMHC-1b/E-restricted CD8⁺ T cells are generated by ex vivo or in vivoinduction of HLA-1a-deprived dendritic cells.

In one embodiment, the CD8 vaccine elicits or comprises suppressorMHC-1b/E-restricted CD8⁺ T cells, wherein the suppressorMHC-1b/E-restricted CD8⁺ T cells are generated by ex vivo or in vivoinduction of HLA-1a-deprived dendritic cells, and wherein theHLA-1a-deprived dendritic cells are obtained by an agent inhibiting TAPexpression or activity.

In another embodiment, the CD8 vaccine is an active vaccine, wherein theCD8 vaccine is a live viral vector comprising at least one HIV antigen,and wherein the live viral vector is selected from the group ofcytomegalovirus, lentivirus, vaccinia virus, adenovirus or plasmid.

In one embodiment, the CD8 vaccine is an active vaccine, and wherein theCD8 vaccine is a cytomegalovirus (CMV) vector comprising:

-   -   a first nucleic acid sequence encoding at least one HIV antigen,    -   optionally a second nucleic acid sequence comprising a first        microRNA recognition element (MRE) operably linked to a CMV gene        that is essential or augmenting for CMV growth, wherein the MRE        silences expression in the presence of a microRNA that is        expressed by a cell of endothelial lineage;        and wherein the CMV vector does not express an active UL128        protein or ortholog thereof; does not express an active UL130        protein or ortholog thereof; does not express an active UL146 or        ortholog thereof; does not express an active UL147 protein or        ortholog thereof,        and wherein the CMV vector expresses at least one active UL40        protein or an ortholog thereof; expresses at least one active        US27 protein or an ortholog thereof and/or expresses at least        one active US28 protein or an ortholog thereof.

In one embodiment, the CD8 vaccine is a cytomegalovirus (CMV) vector,and wherein the CMV vector is a human CMV (hCMV).

In another embodiment, the CD8 vaccine is an active vaccine, and whereinthe CD8 vaccine comprises at least one HIV antigen and a non-pathogenicbacterium.

In one embodiment, the CD8 vaccine comprises at least one HIV antigenand a non-pathogenic bacterium, wherein the HIV antigen is selected fromthe group of virus, virus particles, virus-like particles, recombinantvirus, recombinant virus particles, conjugate viral proteins andconcatemer viral proteins, and wherein said virus, virus particles orsaid recombinant virus particles are attenuated or inactivated.

In one embodiment, the CD8 vaccine comprises at least one HIV antigenand a non-pathogenic bacterium, wherein the non-pathogenic bacterium isliving, and wherein said non-pathogenic bacterium is selected fromattenuated or inactivated pathogenic bacteria.

In one embodiment, the CD8 vaccine comprises at least one HIV antigenand a non-pathogenic bacterium, and wherein the non-pathogenic bacteriumis a Lactobacillus bacterium.

In one embodiment, the CD8 vaccine comprises at least one HIV antigenand a non-pathogenic bacterium, and wherein the non-pathogenic bacteriumis Lactobacillus plantarum.

In another embodiment, the CD8 vaccine is an active vaccine, and whereinthe CD8 vaccine is an ex vivo generated dendritic, natural killer or Bcell population presenting MHC-1b/E-restricted and MHC-II restrictedantigens, and wherein the MHC-1b/E-restricted antigen is an HIV antigen.

In another embodiment, the CD8 vaccine is a passive vaccine, and whereinthe CD8 vaccine is an ex vivo generated autologous MHC-1b/E-restrictedCD8⁺ T cell population, and wherein the MHC-1b/E-restricted CD8⁺ T cellpopulation recognizes an MHC-1b/E-restricted HIV antigen.

In one embodiment, the HIV antigen is derived from any HIV strain, andwherein the HIV antigen is selected from the group consisting of HIVgag, HIV env, HIV rev, HIV tat, HIV nef, HIV pol, and HIV vif antigens.

In one embodiment, the HIV antigen is an HIV-derived HLA-E-bindingantigen.

In one embodiment, the HIV antigen is a HIV-derived HLA-E-bindingantigen, and wherein the HIV-derived HLA-E-binding antigen is selectedfrom the antigens of SEQ ID NO: 1 to SEQ ID NO: 4.

In one embodiment, the method is for preventing acquired immunedeficiency syndrome (AIDS) in a subject in need thereof.

In one embodiment, the method is for prophylactically treating acquiredimmune deficiency syndrome (AIDS) in a subject in need thereof.

In the present invention, the following terms have the followingmeanings:

-   -   “About” preceding a figure encompasses plus or minus 10%, or        less, of the value of said figure. It is to be understood that        the value to which the term “about” refers is itself also        specifically, and preferably, disclosed.    -   As used herein, the term “adjuvant” refers to a compound or        combination of compounds that helps and enhances the        pharmacological effect of a drug or a vaccine, or increases an        immunogenic response, including a CD8⁺ immune response (e.g., an        immune response characterized by a high percentage of the CD8⁺ T        cell response being restricted by MHC-Ib/E used in an infectious        disease treatment).    -   The term “administering” means either directly administering a        compound or composition of the present invention, or        administering a prodrug, derivative or analog which will form an        equivalent amount of the active compound or substance within the        body. For example, according to one embodiment, the fact to        administer a subject with an agent, such as a composition        comprising an effective amount of an HCMV vector comprising an        exogenous antigen by any effective route. Exemplary routes of        administration include, but are not limited to, injection (such        as subcutaneous, intramuscular, intradermal, intraperitoneal,        and intravenous), oral, sublingual, rectal, transdermal,        intranasal, vaginal and inhalation routes.    -   The term “antigen” refers to a compound, composition, or        substance that can stimulate the production of antibodies or a T        cell response in an animal, including compositions that are        injected or absorbed into an animal. An antigen reacts with the        products of specific humoral or cellular immunity, including        those induced by heterologous immunogens. The term “antigen”        includes all related antigenic epitopes. “Epitope” or “antigenic        determinant” refers to a site on an antigen to which B and/or T        cells respond. In one embodiment, T cells respond to the        epitope, when the epitope is presented in conjunction with an        MHC molecule. Epitopes can be formed both from contiguous amino        acids or noncontiguous amino acids juxtaposed by tertiary        folding of a protein. Epitopes formed from contiguous amino        acids are typically retained on exposure to denaturing solvents        whereas epitopes formed by tertiary folding are typically lost        on treatment with denaturing solvents. An epitope typically        includes at least 3, and more usually, at least 5, about 9, or        about 8-10 amino acids in a unique spatial conformation. Methods        of determining spatial conformation of epitopes include, for        example, x-ray crystallography and 2-dimensional nuclear        magnetic resonance. In some embodiments, the antigen is a        pathogen-specific antigen. In the context of the present        disclosure, a pathogen-specific antigen is an antigen that        elicits an immune response against the pathogen and/or is unique        to a pathogen (such as a virus, bacterium, fungus or protozoan).    -   The term “attenuated”, in the context of a live virus or        bacterium, refers to a virus or bacterium with reduced (for        example, eliminated) ability to infect a cell or subject and/or        reduced (for example, eliminated) ability to induce or cause        disease compared to a wild-type virus or wild-type bacterium.        Typically, an attenuated virus or bacterium retains at least        some capacity to elicit an immune response following        administration to an immunocompetent subject. In some cases, an        attenuated virus or bacterium is capable of eliciting a        protective immune response without causing any signs or symptoms        of infection. In some embodiments, the ability of an attenuated        virus or bacterium to cause disease in a subject is reduced at        least about 10%, at least about 25%, at least about 50%, at        least about 75% or at least about 90% relative to wild-type        virus or wild-type bacterium.    -   The term “CMV” (cytomegalovirus) refers to a member of the beta        subclass of the family of herpesviruses. CMV is a large (^(˜)230        kB genome), double stranded DNA virus, with host-range specific        variants such as MCMV (murine CMV), RhCMV (rhesus CMV) and HCMV        (human CMV). In the context of the present invention, “RhCMV”        refers to any strain, isolate or variant of rhesus CMV. In the        context of the present invention, “HCMV” refers to any strain,        isolate or variant of human CMV.    -   The term “decrease” refers to reducing the quality, amount, or        strength of something. For example, a therapy (such as the        methods provided herein) decreases the infectious load or titer        of a pathogen, or one or more symptoms associated with        infection.    -   The term “deletion” refers to the removal of a sequence of DNA,        the regions on either side of the removed sequence being joined        together.    -   The term “expression” refers to the translation of a nucleic        acid into a protein, for example the translation of an mRNA        encoding a tumor-specific or pathogen-specific antigen into a        protein.    -   The term “expression control sequences” refers to nucleic acid        sequences that regulate the expression of a heterologous nucleic        acid sequence to which it is operatively linked, for example the        expression of a heterologous polynucleotide spliced in a CMV        genome and encoding an antigenic protein operably linked to        expression control sequences. Expression control sequences are        operatively linked to a nucleic acid sequence when the        expression control sequences control and regulate the        transcription and, as appropriate, translation of the nucleic        acid sequence. Thus expression control sequences can include        appropriate promoters, enhancers, transcription terminators, a        start codon (ATG) in front of a protein-encoding gene, splicing        signal for introns, and maintenance of the correct reading frame        of that gene to permit proper translation of mRNA, and stop        codons. The term “control sequences” is intended to include, at        a minimum, components whose presence can influence expression,        and can also include additional components whose presence is        advantageous, for example, leader sequences and fusion partner        sequences. Expression control sequences can include a promoter.        A promoter is a minimal sequence sufficient to direct        transcription. Also included are those promoter elements which        are sufficient to render promoter-dependent gene expression        controllable for cell-type specific, tissue-specific, or        inducible by external signals or agents; such elements may be        located in the 5′ or 3′ regions of the gene. Both constitutive        and inducible promoters are included (see for example, Bitter et        al., Methods in Enzymology 153:516-544, 1987). For example, when        cloning in bacterial systems, inducible promoters such as pL of        bacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid        promoter) and the like may be used. In one embodiment, when        cloning in mammalian cell systems, promoters derived from the        genome of mammalian cells (such as metallothionein promoter) or        from mammalian viruses (such as the retrovirus long terminal        repeat; the adenovirus late promoter; the vaccinia virus 7.5K        promoter) can be used. Promoters produced by recombinant DNA or        synthetic techniques may also be used to provide for        transcription of the nucleic acid sequences. A polynucleotide        can be inserted into an expression vector, including a viral        vector, containing a promoter sequence, which facilitates the        efficient transcription of the inserted genetic sequence of the        host. The expression vector typically contains an origin of        replication, a promoter, as well as specific nucleic acid        sequences that allow phenotypic selection of the transformed        cells.    -   The term “fragment” refers to a portion of a polypeptide that        exhibits at least one useful epitope. The phrase “functional        fragment(s) of a polypeptide” refers to all fragments of a        polypeptide that retain an activity, or a measurable portion of        an activity, of the polypeptide from which the fragment is        derived. Fragments, for example, can vary in size from a        polypeptide fragment as small as an epitope capable of binding        an antibody molecule to a large polypeptide capable of        participating in the characteristic induction or programming of        phenotypic changes within a cell. An epitope is a region of a        polypeptide capable of binding an immunoglobulin generated in        response to contact with an antigen.    -   As used herein, the term “heterologous” refers to a heterologous        polypeptide or polynucleotide (such as, for example antigen or a        protein) derived from a different source or species. In some        embodiments of the invention, the heterologous sequence is from        a different genetic source, such as a virus or other organism,        than the second sequence. In particular examples, the        heterologous antigen is not derived from CMV.    -   The term “immunogenic peptide” (or “antigenic peptide”) refers        to a peptide which comprises an allele-specific motif or other        sequence, such as an N-terminal repeat, such that the peptide        will bind an MHC molecule and induce a cytotoxic T lymphocyte        (“CTL”) response, or a B cell response (for example antibody        production) against the antigen from which the immunogenic        peptide is derived. In one embodiment, immunogenic peptides are        identified using sequence motifs or other methods, such as        neural net or polynomial determinations known in the art.        Typically, algorithms are used to determine the “binding        threshold” of peptides to select those with scores that give        them a high probability of binding at a certain affinity and        will be immunogenic. The algorithms are based either on the        effects on MHC binding of a particular amino acid at a        particular position, the effects on antibody binding of a        particular amino acid at a particular position, or the effects        on binding of a particular substitution in a motif-containing        peptide. Within the context of an immunogenic peptide, a        “conserved residue” is one which appears in a significantly        higher frequency than would be expected by random distribution        at a particular position in a peptide. In one embodiment, a        conserved residue is one where the MHC structure may provide a        contact point with the immunogenic peptide.    -   The term “immunity” refers to the state of being able to mount a        protective response upon exposure to an immunogenic agent.        Protective responses can be antibody-mediated or immune        cell-mediated, and can be directed toward a particular pathogen        or tumor antigen Immunity can be acquired actively (such as by        exposure to an immunogenic agent, either naturally or in a        pharmaceutical composition) or passively (such as by        administration of antibodies or in vitro stimulated and expanded        T cells).    -   The term “isolated” or “non-naturally occurring” with reference        to a biological component (such as a nucleic acid molecule,        protein organelle or cells), refers to a biological component        altered or removed from the natural state. For example, a        nucleic acid or a peptide naturally present in a living animal        is not “isolated,” but the same nucleic acid or peptide        partially or completely separated from the coexisting materials        of its natural state is “isolated”. An isolated nucleic acid or        peptide can exist in substantially purified form, or can exist        in a non-native environment such as, for example, a host cell.        Typically, a preparation of isolated nucleic acid or peptide        contains the nucleic acid or peptide at least about 80% pure, at        least about 85% pure, at least about 90% pure, at least about        95% pure, greater than 95% pure, greater than about 96% pure,        greater than about 97% pure, greater than about 98% pure, or        greater than about 99% pure. Nucleic acids and proteins that are        “non-naturally occurring” or have been “isolated” include        nucleic acids and proteins purified by standard purification        methods. The term also embraces nucleic acids and proteins        prepared by recombinant expression in a host cell as well as        chemically synthesized nucleic acids. An “isolated polypeptide”        is one that has been identified and separated and/or recovered        from a component of its natural environment.    -   The terms “individual,” and “patient” are used interchangeably        herein, and refer to an animal, for example a human, to whom        treatment, including prophylactic treatment, with the        pharmaceutical composition according to the present invention,        is provided.    -   The term “subject” as used herein refers to mammals, primates        and/or humans and include all mammals, e.g., mammals, such as        non-human primates, (particularly higher primates), sheep, dog,        rodent, (e.g., mouse or rat), guinea pig, goat, pig, cat,        rabbits, cows, horses.    -   The term “mutation” refers to any difference in a nucleic acid        or polypeptide sequence from a normal, consensus or “wild type”        sequence. A mutant is any protein or nucleic acid sequence        comprising a mutation. In addition a cell or an organism with a        mutation may also be referred to as a mutant. Some types of        coding sequence mutations include point mutations (differences        in individual nucleotides or amino acids); silent mutations        (differences in nucleotides that do not result in an amino acid        changes); deletions (differences in which one or more        nucleotides or amino acids are missing, up to and including a        deletion of the entire coding sequence of a gene); frameshift        mutations (differences in which deletion of a number of        nucleotides indivisible by 3 results in an alteration of the        amino acid sequence. A mutation that results in a difference in        an amino acid may also be called an amino acid substitution        mutation. Amino acid substitution mutations may be described by        the amino acid change relative to wild type at a particular        position in the amino acid sequence.    -   As used herein, an “inactivating mutation” is any mutation in a        viral gene which finally leads to a reduced function or to a        complete loss of function of the viral protein.    -   The term “operably linked” refers to a first nucleic acid        sequence that is operably linked with a second nucleic acid        sequence when the first nucleic acid sequence is placed in a        functional relationship with the second nucleic acid sequence.        For instance, a promoter is operably linked to a coding sequence        if the promoter affects the transcription or expression of the        coding sequence. Generally, operably linked DNA sequences are        contiguous and, where necessary to join two protein-coding        regions, in the same reading frame.    -   The term “open reading frame” (ORF) refers to a series of        nucleotide triplets (codons) coding for amino acids without any        internal termination codons. These sequences are usually        translatable into a peptide.    -   As used herein, the terms “prevent”, “preventing” and        “prevention” refer to preventative measures, wherein the object        is to reduce the chances that a subject will develop the        pathologic condition or disorder over a given period of time.        Such a reduction may be reflected, e.g., in a delayed onset of        at least one symptom of the pathologic condition or disorder in        the subject.    -   The term “prophylactic” refers to a treatment administered to a        subject who does not exhibit signs of a disease or exhibits only        early signs for the purpose of decreasing the risk of developing        pathology. In particular, a prophylactic treatment of a HIV or        SIV infection in a subject refers to a treatment that allows the        subject to become an elite controller (EC) i.e. to have a        relatively high CD4⁺ T cell count (such as e.g. superior to 500        CD4⁺ T cells per microliter) and/or to maintain clinically        undetectable plasma HIV-1 RNA level (such as e.g. HIV RNA <50        copies/mL) during a prolonged period of time in the absence of        any antiretroviral treatment (ART).    -   The term “therapeutic” refers to a treatment administered to a        subject who exhibit early or established signs of a disease.    -   The term “curative” refers to a treatment administered to a        subject suffering from a disease for the purpose of curing the        disease, i.e. of making any sign of the disease disappear or        becoming undetectable.    -   The term “polynucleotide” refers to a polymer of ribonucleic        acid (RNA) or deoxyribonucleic acid (DNA). A polynucleotide is        made up of four bases; adenine, cytosine, guanine, and        thymine/uracil (uracil is used in RNA). A coding sequence from a        nucleic acid is indicative of the sequence of the protein        encoded by the nucleic acid.    -   The terms “protein”, “peptide”, “polypeptide”, and “amino acid        sequence” are used interchangeably herein to refer to polymers        of amino acid residues of any length. The polymer can be linear        or branched, it may comprise modified amino acids or amino acid        analogs, and it may be interrupted by chemical moieties other        than amino acids. The terms also encompass an amino acid polymer        that has been modified naturally or by intervention; for example        disulfide bond formation, glycosylation, lipidation,        acetylation, phosphorylation, or any other manipulation or        modification, such as conjugation with a labeling or bioactive        component.    -   The term “purified” does not require absolute purity; rather, it        is intended as a relative term. Thus, for example, a purified        protein preparation is one in which the protein referred to is        purer than the protein in its natural environment within a cell        or within a production reaction chamber (as appropriate).    -   The term “recombinant” refers to a nucleic acid that has a        sequence that is not naturally occurring or has a sequence that        is made by an artificial combination of two otherwise separated        segments of sequence. This artificial combination can be        accomplished by chemical synthesis or, more commonly, by the        artificial manipulation of isolated segments of nucleic acids,        e.g., by genetic engineering techniques.    -   The term “sample” or “biological sample” refers to a biological        specimen obtained from a subject, such as a cell, fluid of        tissue sample. In some cases, biological samples contain genomic        DNA, RNA (including mRNA and microRNA), protein, or combinations        thereof. Examples of samples include, but are not limited to,        saliva, blood, serum, urine, spinal fluid, tissue biopsy,        surgical specimen, cells (such as PBMCs, white blood cells,        lymphocytes, or other cells of the immune system) and autopsy        material.    -   The term “sequence identity” refers to the similarity between        two nucleic acid sequences, or two amino acid sequences, is        expressed in terms of the similarity between the sequences, and        otherwise referred to as sequence identity. Sequence identity is        frequently measured in terms of percentage identity (or        similarity or homology); the higher the percentage, the more        similar the two sequences are.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman (Adv. Appl. Math. 2: 482, 1981); Needleman and Wunsch (J.Mol. Biol. 48: 443, 1970); Pearson and Lipman (PNAS USA 85: 2444, 1988);Higgins and Sharp (Gene, 73: 237-244, 1988); Higgins and Sharp (CABIOS5: 151-153, 1989); Corpet et al. (Nuc. Acids Res. 16: 10881-10890,1988); Huang et al. (Comp. Appls Biosci. 8: 155-165, 1992); and Pearsonet al. (Meth. Mol. Biol. 24: 307-31, 1994). Altschul et al. (NatureGenet., 6: 119-129, 1994) presents a detailed consideration of sequencealignment methods and homology calculations. The alignment tools ALIGN(Myers and Miller, CABIOS 4:11-17, 1989) or LFASTA (Pearson and Lipman,1988) may be used to perform sequence comparisons (Internet Program©1996, W. R. Pearson and the University of Virginia, fasta20u63 version2.0u63, release date December 1996). ALIGN compares entire sequencesagainst one another, while LFASTA compares regions of local similarity.These alignment tools and their respective tutorials are available onthe Internet at the NCSA Website. Alternatively, for comparisons ofamino acid sequences of greater than about 30 amino acids, the Blast 2sequences function can be employed using the default BLOSUM62 matrix setto default parameters, (gap existence cost of 11, and a per residue gapcost of 1). When aligning short peptides (fewer than around 30 aminoacids), the alignment should be performed using the Blast 2 sequencesfunction, employing the PAM30 matrix set to default parameters (open gap9, extension gap 1 penalties). The BLAST sequence comparison system isavailable, for instance, from the NCBI web site; see also Altschul etal., J. Mol. Biol. 215:403-410, 1990; Gish. & States, Nature Genet.3:266-272, 1993; Madden et al. Meth. Enzymol. 266:131-141, 1996;Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; and Zhang &Madden, Genome Res. 7:649-656, 1997.

Orthologs of proteins are typically characterized by possession ofgreater than 75% sequence identity counted over the full-lengthalignment with the amino acid sequence of specific protein using ALIGNset to default parameters. Proteins with even greater similarity to areference sequence will show increasing percentage identities whenassessed by this method, such as at least 80%, at least 85%, at least90%, at least 92%, at least 95%, or at least 98% sequence identity. Inaddition, sequence identity can be compared over the full length ofparticular domains of the disclosed peptides.

When significantly less than the entire sequence is being compared forsequence identity, homologous sequences will typically possess at least80% sequence identity over short windows of 10-20 amino acids, and maypossess sequence identities of at least 85%, at least 90%, at least 95%,or at least 99% depending on their similarity to the reference sequence.Sequence identity over such short windows can be determined usingLFASTA; methods are described at the NCSA Website. One of skill in theart will appreciate that these sequence identity ranges are provided forguidance only; it is entirely possible that strongly significanthomologs could be obtained that fall outside of the ranges provided.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences, due to the degeneracyof the genetic code. It is understood that changes in nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid sequences that each encode substantially the same protein.

-   -   As used herein, the term “treatment” refers to an intervention        that ameliorates a sign or symptom of a disease or pathological        condition. For example, in case of HIV infection, HIV RNA (viral        load) and CD4 T lymphocyte (CD4) cell count are the two        surrogate markers of antiretroviral treatment (ART) responses        and HIV disease progression that have been used for decades to        manage and monitor HIV infection. Thus, the efficacy of the        treatment may be evaluated by the plasma viral RNA load of a        “treated” human before and after the treatment, if it is reduced        by at least about 10%, 20%, 30%, 40%, 50%, more preferably by at        least about 70%, yet more preferably by at least about 75% or        80% or 85% or 90% or 95% or 98% or 99%, or even more (99.5%,        99.8%, 99.9%, 100%) the treatment is considered as effective,        and/or by the monitoring of CD4 cell count before and after the        treatment, if the absolute count of CD4 cell is increased by at        least about 5%, 10%, 15%, 20%, 25%, more preferably by at least        about 30%, yet more preferably by at least about 35% or 40% or        45% or 50% or 55% or 60% or 65%, or even more the treatment is        considered as effective. As used herein, the terms “treatment”,        “treat” and “treating,” with reference to a disease,        pathological condition or symptom, also refers to any observable        beneficial effect of the treatment. The beneficial effect can be        evidenced, for example, by a delayed onset of clinical symptoms        of the disease in a susceptible subject, a reduction in severity        of some or all clinical symptoms of the disease, a slower        progression of the disease, a reduction in the number of        relapses of the disease, an improvement in the overall health or        well-being of the subject, or by other parameters well known in        the art that are specific to the particular disease. A        therapeutic treatment is a treatment administered to a subject        after signs and symptoms of the disease have developed. A        prophylactic treatment is a treatment administered to a subject        who does not exhibit signs of a disease or exhibits only early        signs, for the purpose of decreasing the risk of developing        pathology. In particular, a prophylactic treatment of a HIV or        SIV infection in a subject refers to a treatment that allows the        subject to become an elite controller (EC) i.e. to have a        relatively high CD4⁺ T cell count (such as e.g. superior to 500        CD4⁺ T cells per microliter) and/or to maintain clinically        undetectable plasma HIV-1 RNA level (such as e.g. HIV RNA <50        copies/mL) during a prolonged period of time in the absence of        any antiretroviral treatment (ART). A prophylactic treatment is        a treatment administered to a subject suffering from a disease        for the purpose of curing the disease, i.e. of making any sign        of the disease disappear or becoming undetectable.    -   The term “vector” may include nucleic acid sequences that permit        it to replicate in a host cell, such as an origin of        replication. A vector may also include one or more selectable        marker genes and other genetic elements known in the art,        including promoter elements that direct nucleic acid expression.        Vectors can be viral vectors, such as CMV vectors. Viral vectors        may be constructed from wild type or attenuated virus, including        replication deficient virus. Vectors can also be non-viral        vectors, including any plasmid known to the art.    -   The term “virus” refers to microscopic infectious organism that        reproduces inside living cells. A virus consists essentially of        a core of nucleic acid (the viral genome) surrounded by a        protein coat (capsid), and has the ability to replicate only        inside a living cell. “Viral replication” is the production of        additional virus particles by the occurrence of at least one        viral life cycle. A virus may subvert the host cells' normal        functions, causing the cell to behave in a manner determined by        the virus. For example, a viral infection may result in a cell        producing a cytokine, or responding to a cytokine, when the        uninfected cell does not normally do so. The term “lytic” or        “acute” viral infection refers to a viral infection wherein the        viral genome is replicated and expressed, producing the        polypeptides necessary for production of the viral capsid.        Mature viral particles exit the host cell, resulting in cell        lysis. Particular viral species can alternatively enter into a        “lysogenic” or “latent” infection. In the establishment of        latency, the viral genome is replicated, but capsid proteins are        not produced and assembled into viral particles.    -   As used herein, the term “microRNA” or “miRNA” refers to a major        class of biomolecules involved in control of gene expression.        For example, in human heart, liver or brain, miRNAs play a role        in tissue specification or cell lineage decisions. In addition,        miRNAs influence a variety of processes, including early        development, cell proliferation and cell death, and apoptosis        and fat metabolism. The large number of miRNA genes, the diverse        expression patterns, and the abundance of potential miRNA        targets suggest that miRNAs may be a significant source of        genetic diversity.    -   A mature miRNA is typically an 18-25 nucleotide non-coding RNA        that regulates expression of an mRNA including sequences        complementary to the miRNA. These small RNA molecules are known        to control gene expression by regulating the stability and/or        translation of mRNAs. For example, miRNAs bind to the 3′ UTR of        target mRNAs and suppress translation. MiRNAs may also bind to        target mRNAs and mediate gene silencing through the RNAi        pathway. MiRNAs may also regulate gene expression by causing        chromatin condensation.    -   A miRNA silences translation of one or more specific mRNA        molecules by binding to a miRNA recognition element (MRE), which        is defined as any sequence that directly base pairs with and        interacts with the miRNA somewhere on the mRNA transcript.        Often, the MRE is present in the 3′ untranslated region (UTR) of        the mRNA, but it may also be present in the coding sequence or        in the 5′ UTR. MREs are not necessarily perfect complements to        miRNAs, usually having only a few bases of complementarity to        the miRNA and often containing one or more mismatches within        those bases of complementarity. The MRE may be any sequence        capable of being bound by a miRNA sufficiently that the        translation of a gene to which the MRE is operably linked (such        as a CMV gene that is essential or augmenting for growth in        vivo) is repressed by a miRNA silencing mechanism such as the        RISC.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A. Antiviral activity of type I and type III interferons.Expression of ISGs in HepG2. HepG2 cells were treated with IFNα2a orIFNκ1-4 (10 ng/ml). After 4 h of stimulation, qRT-PCR were used toexamine the mRNA levels of the interferon-induced genes, IFIT1, MX1 andOASL and fold-changes was calculated by 2-ΔΔCt method as compared withnon-treated cell control and using endogenous S14 mRNA level fornormalization.

FIG. 1B. Antiviral activity of type I and type III interferons.Antiviral activity of type I and III IFNs against EMCV. IFNα2a orIFNλ1/2/3/4 (10 ng/ml) were added to HepG2 cells 24 h prior to challengewith EMCV. Forty-eight after infection with EMCV, cells were assayed forviability with a bioassay. A570 values were directly proportional tocell viability and therefore antiviral activity of the respective IFNs.IFN-α treatment without viral challenge was used as a baseline of theviability of the cells.

FIG. 2. Anti-proliferative activity of type I and type III interferonsagainst CD4⁺ T cells. CFSE-stained CD4⁺ T cells (10×10⁴/well) werestimulated for 5 days in 96 round-bottomed microwells with allogeneicpoly I:C matured DC in absence (control) or presence of 10 ng/ml ofIFN-α2a, or IFNλ1 or IFλ2 or IFNλ3 or IFNλ4. When indicated,anti-interferon type I receptor antibody was added. The percentage ofCFSE dilution was evaluated by flow cytometry.

FIG. 3. IFN-α2a but not IFN-type III induces the expression of ISGs inCD4⁺ T cells. CD4⁺ T cells were treated with IFNα2a or IFNλ1/2/3/4 (10ng/ml). After 4 h of stimulation, qRT-PCR were used to examine the mRNAlevels of the interferon-induced genes, IFIT1, MX1 and OASL andfold-changes was calculated by 2^(−ΔΔCt) method as compared withnon-treated cell control and using endogenous S14 mRNA level fornormalization.

FIG. 4. IFN-α2a but not IFN-type III stimulates the phosphorylation ofStat1 in CD4⁺ T cells. CD4⁺ T cells were stimulated with 10 ng ml⁻¹ ofIFN-λ1, IFN-λ2, IFN-λ3, IFN-λ4, or IFN-α2a for 20 min, or were leftunstimulated (control). Increases in pSTAT1 were evaluated as a ratio ofinduction over baseline levels (MFI fold change=MFIcytokine-stimulated/MFI untreated cells)

FIG. 5. IFN-α2a but not IFN-type III increases CD38 expression inCD3/CD28 stimulated CD4⁺ T cells. CFSE-stained CD4⁺ T cells (4×10⁴/well)were cultured in 96 round-bottomed microwells in the presence ofΔCD3-feeder (4×10⁴/well) and plate-bound anti-CD3 mAb (2 μg/ml), solubleanti-CD28 mAb (2 μg/ml) with increasing dose of IFN-α2a or IFN type III.CD38 Median Fluorescence Intensity (MFI) was measured by flow cytometryin CD3⁺ 7-AAD-CFSE⁺ stimulated CD4⁺ T cells at the end of the culture.

FIG. 6. Generation and expansion of peptide-specific CD8 HLA-Erestricted by peptide-loaded m-DCs. TAP-inhibited mDCs was pulsed withpeptides (10 μM for 1 h) and co-cultured with autologous naive CD8⁺ Tcells at a 1:10 ratio. Peptide positive CD8⁺ T cells was monitored atday 0 and one week after the last stimulation by flow cytometricanalysis using MHC-peptide pentamers. Data are expressed as percentageof tetramer-positive cells among CD8⁺ T cells.

FIG. 7. Schematic representation of the immunization protocol inmacaques with RhCMV/SIV vectors.

FIG. 8. Schematic representation of the oral immunization protocol inmacaques with inactivated SIV and living Lactobacillus plantarum.

FIG. 9. Schematic representation of the oral immunization protocol inBLT mice with inactivated HIV and living Lactobacillus plantarum.

FIG. 10A. Comparison of CM CD8⁺ T cell distributions and serum IFN-αlevels in HIV-1-infected subjects and critical pathogenic role of IFN-αin human HIV-1 infection. Comparison of CM CD8⁺ T cell distributions inHIV-1-infected subjects.

FIG. 10B. Comparison of CM CD8⁺ T cell distributions and serum IFN-αlevels in HIV-1-infected subjects and critical pathogenic role of IFN-αin human HIV-1 infection. Comparison of serum IFN-α levels inHIV-1-infected subjects.

FIG. 10C. Comparison of CM CD8⁺ T cell distributions and serum IFN-αlevels in HIV-1-infected subjects and critical pathogenic role of IFN-αin human HIV-1 infection. Relationship between CD8+CM frequency andserum IFN-α level in non-treated HIV patients (EC and pre-cART group).

FIG. 11. Schematic representation of the oral immunization protocol inmacaques with inactivated SIV and living Lactobacillus plantarum.

FIG. 12. Schematic representation of the oral immunization protocol inBSLT mice with inactivated HIV and living Lactobacillus plantarum.

DETAILED DESCRIPTION

This invention relates to a method for preventing or treating aninfectious disease in a subject in need thereof, comprisingadministering to the subject:

-   -   1) a CD8 vaccine specific for at least one infectious        disease-related antigen,    -   2) an interferon alpha blocking agent, and/or    -   3) a type III interferon and/or an agent stimulating the        production of type III interferon.

In one embodiment, the method comprises administering to the subject 1)a CD8 vaccine specific for at least one infectious disease-relatedantigen, 2) an interferon alpha blocking agent, and 3) a type IIIinterferon and/or an agent stimulating the production of type IIIinterferon.

In another embodiment, the method comprises administering to thesubject 1) a CD8 vaccine specific for at least one infectiousdisease-related antigen and 2) an interferon alpha blocking agent.

In another embodiment, the method comprises administering to thesubject 1) a CD8 vaccine specific for at least one infectiousdisease-related antigen and 3) a type III interferon and/or an agentstimulating the production of type III interferon.

In some embodiments, the method is a method of prophylactic treatment ora method of curative treatment.

In a particular embodiment, the method is a prophylactic method andcomprises administering to the subject 1) a CD8 vaccine specific for atleast one infectious disease-related antigen, 2) an interferon alphablocking agent, and 3) a type III interferon and/or an agent stimulatingthe production of type III interferon.

The present invention further relates to a combination for use as amedicament, wherein said combination comprises:

-   -   1) a CD8 vaccine specific for at least one infectious        disease-related antigen,    -   2) an interferon alpha blocking agent, and/or    -   3) a type III interferon and/or an agent stimulating the        production of type III interferon.

The present invention also relates to a combination for use in theprevention or treatment of an infectious disease in a subject in needthereof, wherein said combination comprises:

-   -   1) a CD8 vaccine specific for at least one infectious        disease-related antigen,    -   2) an interferon alpha blocking agent, and/or    -   3) a type III interferon and/or an agent stimulating the        production of type III interferon.

In one embodiment, the combination for use comprises 1) a CD8 vaccinespecific for at least one infectious disease-related antigen, 2) aninterferon alpha blocking agent, 3) and a type III interferon and/or anagent stimulating the production of type III interferon.

In one embodiment, the combination for use comprises 1) a CD8 vaccinespecific for at least one infectious disease-related antigen and 2) aninterferon alpha blocking agent.

In one embodiment, the combination for use comprises 1) a CD8 vaccinespecific for at least one infectious disease-related antigen and 3) atype III interferon and/or an agent stimulating the production of typeIII interferon.

In a particular embodiment, the combination is to be usedprophylactically and comprises 1) a CD8 vaccine specific for at leastone infectious disease-related antigen, 2) an interferon alpha blockingagent, and 3) a type III interferon and/or an agent stimulating theproduction of type III interferon.

The present invention further relates to a kit-of-parts for use in theprevention or treatment of an infectious disease in a subject in needthereof, wherein said kit-of-parts comprises at least 2 parts, andcomprises:

-   -   1) a first part comprising a CD8 vaccine specific for at least        one infectious disease-related antigen,    -   2) optionally a second part comprising an interferon-alpha        blocking agent, and    -   3) a third part comprising a type III interferon and/or an agent        stimulating the production of type III interferon.

As used herein, the term “vaccine” refers to an immunogenic product orcomposition that can be administered to a mammal, such as a human, toconfer immunity, such as passive or active immunity, to a disease orother pathological condition. Vaccines can be used preventively ortherapeutically, either prophylactically or curatively. Thus, vaccinescan be used to reduce the likelihood of developing a disease (such asinfection) or to reduce the severity of symptoms of a disease orcondition, limit the progression of the disease or condition (such asinfection), or limit the recurrence of a disease or condition.

In one embodiment, the CD8 vaccine is a preventive vaccine. In anotherembodiment, the CD8 vaccine is a therapeutic vaccine. As used herein, atherapeutic vaccine may be a prophylactic vaccine or a curative vaccine.In some embodiments, the CD8 vaccine is a prophylactic vaccine. In otherembodiments, the CD8 vaccine is a curative vaccine.

In one embodiment, the CD8 vaccine induces immunotolerance to at leastone infectious disease-related antigen. In one embodiment, the CD8vaccine is thus specific for at least one infectious diseases-relatedantigen.

As used herein, the terms “immunotolerance” and “Ts” are synonymous.Immunotolerance is the physiological capacity of the immune system torecognize antigens and to develop anergy generally associated with otherimmunological modifications to a subsequent encounter with the sameantigens. In the present invention, immunotolerance is principallycharacterized by the activity of CD8⁺ T cells which suppress theactivation CD4⁺ T cells that present at least one infectiousdiseases-related antigen. Globally, each time where one or severalinfectious diseases-related antigen is/are involved in the specificactivation of CD4⁺ T cells (which present epitopes derived from theinfectious diseases-related antigen), the specificsuppression/prevention of activation of CD4⁺ T cell can be raised byMHC-1b/E-restricted CD8⁺ T cells generated by the CD8 vaccines asdescribed in the present invention.

In one embodiment, the CD8 vaccine of the invention elicits suppressorMHC-1b/E-restricted CD8⁺ T cells. In another embodiment, the CD8 vaccineof the invention comprises or consist essentially of suppressorMHC-1b/E-restricted CD8⁺ T cells.

As used herein, “consisting essentially of”, with reference to a cellpopulation, means that the suppressor MHC-1b/E-restricted CD8⁺ T cellpopulation is the only one therapeutic agent or agent with a biologicactivity within said composition.

In one embodiment, the suppressor MHC-1b/E-restricted CD8⁺ T cells aregenerated by ex vivo or in vivo induction of HLA-1a-deprived dendritic,natural killer or B cells.

In one embodiment, the suppressor MHC-1b/E-restricted CD8⁺ T cells arecytolytic CD8⁺ T cells. In one embodiment, the suppressorMHC-1b/E-restricted CD8⁺ T cells are non-cytolytic CD8⁺ T cells.

In one embodiment, the CD8 vaccine is an active vaccine. In anotherembodiment, the CD8 vaccine is a passive vaccine.

As used herein, the term “active vaccine” refers to a vaccine thatinduces an active immunity and refers to the process of exposing thebody to an antigen to generate an adaptive immune response: the responsetakes days/weeks to develop but may be long lasting—even lifelong. A“passive vaccine” induces a passive immunity and refers to the processof providing, for example, antibodies or cells to protect againstinfection; it gives immediate, but short-lived protection—several weeksto months.

In some embodiments, the CD8 vaccine comprises suppressorMHC-1b/E-restricted CD8⁺ T cells.

In some embodiments, the CD8 vaccine is a vaccine eliciting suppressorMHC-1b/E-restricted CD8⁺ T cells, said vaccine being selected from thegroup consisting of:

-   -   an active vaccine which is a live viral vector comprising at        least one pathogen-specific antigen, wherein the live viral        vector is selected from the group consisting of cytomegalovirus,        lentivirus, vaccinia virus, adenovirus and plasmid;    -   an active vaccine comprising at least one pathogen-specific        antigen and at least one non-pathogenic bacterium, preferably at        least one attenuated or inactivated pathogenic bacterium;    -   an active vaccine which is an ex vivo generated dendritic,        natural killer or B cell population presenting at least one        MHC-1b/E-restricted antigen and at least one MHC-II restricted        antigen, and wherein the MHC-1b/E-restricted antigen is a        pathogen-specific antigen.    -   a passive vaccine which is an ex vivo generated autologous        MHC-1b/E-restricted CD8⁺ T cell population, recognizing an        MHC-1b/E-restricted pathogen-specific antigen.

According to one embodiment of the invention, the CD8 vaccine is a liveviral vector comprising at least one infectious diseases-relatedantigen.

In one embodiment, the live viral vector, as described hereinabove, isan active vaccine.

In one embodiment, the live viral vector, as described hereinabove, isselected from the group of cytomegalovirus, lentivirus, vaccinia virus,adenovirus and plasmid.

In one embodiment, the live viral vector, as described hereinabove, is arecombinant vector selected from the group of recombinantcytomegalovirus, recombinant lentivirus, recombinant vaccinia virus,recombinant adenovirus and recombinant plasmid.

According to one embodiment, the live viral vector is a recombinantvaccinia virus. Recombinant vaccinia viruses have been produced fromdifferent Vaccinia virus strains. For example, a variety of highlyattenuated, host-restricted, non- or poorly-replicating poxvirus strainsfor use as substrates in recombinant vaccine development have beendeveloped, including the Orthopoxviruses, Modified Vaccinia Ankara(MVA), NYVAC, Avipoxviruses, ALVAC, and TROVAC.

According to one embodiment of the invention, the CD8 vaccine is a CMVvector.

In one embodiment, the CMV vector is an active vaccine.

In one embodiment, the CMV vector comprises a nucleic acid sequence thatencodes at least one infectious disease-related antigen. In a particularembodiment, the CMV vector comprises a nucleic acid sequence thatencodes at least one human immunodeficiency virus (HIV) antigen.

In one embodiment, the CD8 vaccine is a recombinant CMV expressing atleast one infectious diseases-related antigen, wherein said antigen is aheterologous antigen. Thus, in one embodiment, the infectiousdiseases-related antigen can be derived from any protein that is notnatively expressed in CMV.

In one embodiment, the CMV vector does not express an active UL128 andUL130 proteins, or orthologs thereof.

As used herein, the term “ortholog” refers to homologous genes of CMVsthat infect other species.

In one embodiment, the CMV vector does not express an active UL146 andUL147 proteins, or orthologs thereof.

In one embodiment, the CMV vector expresses at least one active UL40protein, and/or at least one active US27 protein, and/or at least oneactive US28 protein. In one embodiment, the at least one active UL40protein, the at least one active US27 and the at least one active US28protein can be orthologs or homologs of UL40, US27 and US28.

In some examples, the CMV vector does not express an active UL128,UL130, UL146 or UL147 protein due to the presence of a mutation in thenucleic acid sequence encoding UL128, UL130, UL146 or UL147, ororthologs thereof.

As used herein, the term “mutation” may refer to any mutation thatresults in a lack of expression of active UL128, UL130, UL146 or UL147protein. Such mutations can include point mutations, frameshiftmutations, deletions of less than all of the sequence that encodes theprotein (truncation mutations), or deletions of all of the nucleic acidsequence that encodes the protein, or any other mutations. For example,CMV comprising said mutation are described in WO2014138209, which isincorporate by reference herein in its entirety.

In further examples, the vector does not express an active UL128, UL130,UL146 or UL147 protein, or an ortholog thereof, due to the presence of anucleic acid sequence in the vector that comprises an antisense or RNAisequence (siRNA or miRNA) that inhibits the expression of the UL128,UL130, UL146 or UL147 protein, or an ortholog thereof.

In one embodiment, mutations and/or antisense and/or RNAi can be used inany combination to generate a CMV vector lacking active UL128, UL130,UL146 or UL147, or an ortholog thereof.

In one embodiment, the CMV vectors comprises all of the abovemodifications and further comprises a nucleic acid sequence that servesas a miRNA response element (MRE) that silences expression in thepresence of a miRNA expressed by endothelial cells.

As used herein, the term “miRNA response element” or “MRE” refers to anysequence that directly base pairs with and interacts with the miRNAsomewhere on the mRNA transcript. Thus, a miRNA may silence thetranslation of one or more specific mRNA molecules by binding to a miRNArecognition element (MRE). Often, the MRE is present in the 3′untranslated region (UTR) of the mRNA, but it may also be present in thecoding sequence or in the 5′ UTR. MREs are not necessarily perfectlycomplementary to miRNAs, usually having only a few bases ofcomplementarity to the miRNA and often containing one or more mismatcheswithin those bases of complementarity. The MRE may be any sequencecapable of being bound by a miRNA sufficiently that the translation of agene to which the MRE is operably linked. Examples of such genes includewithout limitation IE2 and UL79 genes, or orthologs thereof, or any CMVgene that is essential or augmenting for growth in vivo. For example,CMV comprising said MRE are described in WO201875591, which isincorporate by reference herein in its entirety.

In one embodiment, the MRE may be any miRNA recognition element thatsilences expression in the presence of a miRNA expressed by endothelialcells. In one embodiment, an MRE of the vector silences expression inthe presence of one or more of miR-126-3p, miR-130a, miR-210,miR-221/222, miR-378, miR-296, and miR-328.

In one embodiment, the MRE silences expression in the presence ofmiR-126-3p.

In one embodiment, the MRE silences the expression of UL122 (IE2) andUL79 in the presence of miR-126-3p.

One of skill in the art may select a validated, putative, or mutated MREsequence from the literature that would be predicted to induce silencingin the presence of a miRNA expressed in an endothelial cell or a myeloidcell such as a macrophage. The person of skill in the art may thenobtain an expression construct whereby a reporter gene (such as afluorescent protein, enzyme or other reporter gene) has expressiondriven by a promoter such as a constitutively active promoter orcell-specific promoter. The MRE sequence may then be introduced into theexpression construct. The expression construct may be transfected intoan appropriate cell, and the cell transfected with the miRNA ofinterest. A lack of expression of the reporter gene indicates that theMRE silences gene expression in the presence of the miRNA.

In one embodiment, the CMV vector comprises a first nucleic acidsequence encoding at least one infectious diseases-related antigen, anddoes not express an active UL128, UL130, UL146 and UL147 proteins ororthologs thereof, and expresses at least one active UL40, US27 and/orUS28 proteins or an orthologs thereof.

In another embodiment, the CMV vector comprises a first nucleic acidsequence encoding at least one infectious diseases-related antigen,optionally a second nucleic acid sequence comprising a first microRNArecognition element (MRE) operably linked to a CMV gene that isessential or augmenting for CMV growth, wherein the MRE silencesexpression in the presence of a microRNA that is expressed by a cell ofendothelial lineage, and does not express an active UL128, UL130, UL146and UL147 proteins or orthologs thereof, and expresses at least oneactive UL40, US27 and/or US28 proteins or an orthologs thereof.

In one embodiment, the CMV vector can comprise additional inactivatingmutations known in the art to provide different immune responses, suchas an inactivating US11 mutation or an inactivating UL82 (pp71)mutation, or any other inactivating mutation.

In one embodiment, the CMV vector may also comprise at least oneinactivating mutations in one or more viral genes encoding viralproteins known in the art to be essential or augmenting for viraldissemination (i.e., spread from cell to cell) in vivo. Suchinactivating mutations may result from point mutations, frameshiftmutations, truncation mutations, or a deletion of all of the nucleicacid sequence encoding the viral protein. Inactivating mutations includeany mutation in a viral gene which finally leads to a reduced functionor to a complete loss of function of the viral protein.

In one embodiment, the CMV vectors described herein can containmutations that can prevent host to host spread, thereby rendering thevirus unable to infect immunocompromised or other subjects that couldface complications as a result of CMV infection. In another embodiment,the CMV vectors described herein can also contain mutations that resultin the presentation of immunodominant and non-immunodominant epitopes aswell as non-canonical MHC restriction. Such CMV mutations are describedin, for example, US Patent Publications 2013-0136768; 2014-0141038; andPCT application publication WO 2014/138209, all of which areincorporated by reference herein.

In one embodiment, mutations in the CMV vectors described herein do notaffect the ability of the vector to re-infect a subject that has beenpreviously infected with CMV. Accordingly, in one embodiment, the CMVvector is capable of repeatedly infecting an organism.

In one embodiment, the CMV vector is a human CMV (hCMV) or rhesus CMV(RhCMV) vector.

In on embodiment, the CMV vectors disclosed herein can be prepared byinserting DNA comprising a sequence that encodes the infectiousdisease-related antigen into an essential or non-essential region of theCMV genome.

In one embodiment, the infectious disease-related antigen is aheterologous antigen of the CMV.

In one embodiment, the method can further comprise deleting one or moreregions from the CMV genome. In one embodiment, the method can comprisein vivo recombination. Thus, the method can comprise transfecting a cellwith CMV DNA in a cell-compatible medium in the presence of donor DNAcomprising the heterologous DNA flanked by DNA sequences homologous withportions of the CMV genome, whereby the heterologous DNA is introducedinto the genome of the CMV, and optionally then recovering CMV modifiedby the in vivo recombination.

In one embodiment, the method can also comprise cleaving CMV DNA toobtain cleaved CMV DNA, ligating the heterologous DNA to the cleaved CMVDNA to obtain hybrid CMV-heterologous DNA, transfecting a cell with thehybrid CMV-heterologous DNA, and optionally then recovering CMV modifiedby the presence of the heterologous DNA. Since in vivo recombination iscomprehended, the method accordingly also provides a plasmid comprisingdonor DNA not naturally occurring in CMV encoding a polypeptide foreignto CMV, the donor DNA is within a segment of CMV DNA that wouldotherwise be co-linear with an essential or non-essential region of theCMV genome such that DNA from an essential or nonessential region of CMVis flanking the donor DNA. The heterologous DNA can be inserted into CMVto generate the recombinant CMV in any orientation that yields stableintegration of that DNA, and expression thereof, when desired.

In one embodiment, the DNA encoding the infectious disease-relatedantigen in the recombinant CMV vector can also include a promoter. Thepromoter can be from any source such as a herpes virus, including anendogenous CMV promoter, such as a HCMV, RhCMV, murine CMV (MCMV), orother CMV promoter. The promoter can also be a non-viral promoter suchas the EF1a promoter. The promoter can be a truncated transcriptionallyactive promoter which comprises a region transactivated with atransactivating protein provided by the virus and the minimal promoterregion of the full-length promoter from which the truncatedtranscriptionally active promoter is derived. The promoter can becomposed of an association of DNA sequences corresponding to the minimalpromoter and upstream regulatory sequences. A minimal promoter iscomposed of the CAP site plus TATA box (minimum sequences for basiclevel of transcription; unregulated level of transcription); “upstreamregulatory sequences” are composed of the upstream element(s) andenhancer sequence(s). Further, the term “truncated” indicates that thefull-length promoter is not completely present, i.e., that some portionof the full-length promoter has been removed. And, the truncatedpromoter can be derived from a herpesvirus such as MCMV or HCMV, e.g.,HCMV-IE or MCMV-IE. There can be up to a 40% and even up to a 90%reduction in size, from a full-length promoter, based upon base pairs.The promoter can also be a modified non-viral promoter. As to HCMVpromoters, reference is made to U.S. Pat. Nos. 5,168,062 and 5,385,839which are incorporated herein by reference. As to transfecting cellswith plasmid DNA for expression therefrom, reference is made to Feigneret al. (1994), J. Biol. Chem. 269, 2550-2561 which is incorporatedherein by reference. And, as to direct injection of plasmid DNA as asimple and effective method of vaccination against a variety ofinfectious diseases reference is made to Ulmer et al. (1993), Science.259:1745-49, which is incorporated herein by reference. It is thereforewithin the scope of this invention that the vector can be used by thedirect injection of vector DNA.

Also disclosed is an expression cassette that can be inserted into arecombinant virus or plasmid comprising the truncated transcriptionallyactive promoter. The expression cassette can further include afunctional truncated polyadenylation signal; for instance an SV40polyadenylation signal which is truncated, yet functional. Consideringthat nature provided a larger signal, it is indeed surprising that atruncated polyadenylation signal is functional. A truncatedpolyadenylation signal addresses the insert size limit problems ofrecombinant viruses such as CMV. The expression cassette can alsoinclude heterologous DNA with respect to the virus or system into whichit is inserted; and that DNA can be heterologous DNA as describedherein.

For the disclosed infectious disease-related antigen to be expressed inthe vector, the protein coding sequence of the infectiousdisease-related antigen should be “operably linked” to regulatory ornucleic acid control sequences that direct transcription and translationof the protein.

According to another embodiment of the invention, the CD8 vaccinecomprises at least one infectious disease-related antigen and anon-pathogenic bacterium.

In one embodiment, the CD8 vaccine comprises at least one infectiousdisease-related antigen and at least one non-pathogenic bacterium.

In one embodiment, the CD8 vaccine that comprises at least oneinfectious disease-related antigen and a non-pathogenic bacterium is anactive vaccine.

As used herein, the term “non-pathogenic bacterium” refers to bacteriathat do not generally induce any pathology in mammals, preferably inhumans

In one embodiment, the non-pathogenic bacterium is living.

In one embodiment, the non-pathogenic bacterium described herein is acommensal bacterium.

As used herein, the term “commensal bacterium” or refers tomicro-organism, which is present on body surfaces covered by epithelialcells and is exposed to the external environment (e.g., gastrointestinaland respiratory tract, vagina, skin, etc.). Among the numerous proposedhealth benefits attributed to intestinal commensal bacteria, theircapacity to interact with the host immune system is now welldemonstrated. Commensal bacteria are well-known to the skilled artisan.Non-limiting examples include Bacillus sp. (e.g., B. coagulans),Lactobacillus sp., Bifidobacterium animalis, Bifidobacterium breve,Bifidobacterium infantis, Bifidobacterium longum, Bifidobacteriumbifidum, Bifidobacterium lactis, Escherichia coli, Lactobacillusacidophilus, Lactobacillus bulgaricus, Lactobacillus casei,Lactobacillus paracasei, Lactobacillus johnsonii, Lactobacillusplantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillusbrevis, Lactobacillus gasseri, Lactobacillus salivarius, Lactobacillussalivarius salicinius, Lactobacillus delbureckii, Lactobacillusdelbureckii bulgaricus, Lactobacillus delbureckii lactis, Lactococcuslactis, Streptococcus thermophilus, and the like.

In one embodiment, the commensal bacterium is selected from the group ofLactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillusplantarum, Bifidobacterium bifidum, Bifidobacterium breve, Lactococcuslactis, Streptococcus thermophilus, Lactobacillus casei, Lactobacillusacidophilus, Lactobacillus reuteri.

In one embodiment, the commensal bacterium is Lactobacillus sp.,preferably Lactobacillus plantarum.

In another embodiment, the commensal bacterium is Lactobacillus sp.,preferably Lactobacillus rhamnosus.

In one embodiment, a combination of non-pathogenic bacteria, such as twoor more commensal bacteria, may be used.

In another embodiment, the non-pathogenic bacterium described herein isselected from attenuated or inactivated pathogenic bacteria.

As used herein, the terms “pathogenic bacteria” refer to bacteriainducing pathologies in humans. Such bacteria are well known from theskilled person and include inter alia Listeria species (e.g., Listeriamonocytogenes), Corynebacterium species, Mycobacterium species,Rhococcus species, Eubacteria species, Bortadella species and Nocardiaspecies. Preferably, a pathogenic bacterium is selected amongMycobacterium species, and is more preferably Mycobacterium bovis.

As used herein, the terms “attenuated pathogenic bacteria” refer tobacteria which are less virulent compared to their wild-type counterpartbecause of one or several mutations or of one or more attenuationtreatments (e.g., chemical treatment and/or successive passages onspecific media). Such attenuated pathogenic bacteria are well known fromthe one of skill in the art. Non-limiting examples of attenuatedpathogenic bacteria include attenuated Salmonella typhimurium andMycobacteria. Methods of preparation of such inactivated pathogenicbacteria form part of the common general knowledge in the art. As anexample of such methods, one can cite phage mediated lysis, chemicalinactivation such as formalin treatment, thermal inactivation, physicalinactivation such as lyophilisation (e.g., Extended Freeze Drying) orU.V or gamma irradiation or microwave exposure, or any combinationthereof.

In one embodiment, the non-pathogenic bacterium described herein may berecombinant or not.

In one embodiment, the attenuated pathogenic bacterium described hereinis an attenuated derivative of pathogenic bacteria like BCG. In oneembodiment, said attenuated derivative of pathogenic bacteriacorresponds to recombinant Salmonella typhimurium or recombinantMycobacteria (e.g., BCG) which expresses or produces at least one HIVprotein. In another embodiment, said derivative of pathogenic bacteriadoes not express any HIV protein.

In one embodiment, the non-pathogenic bacterium described herein is tobe used as a tolerogenic adjuvant of the CD8 vaccine. Accordingly, inone embodiment, the non-pathogenic bacterium is a tolerogenic adjuvant.

As used herein, the term “tolerogenic adjuvant” is an entity that, whenadministered by the mucosal or the intradermal or the intraepithelialroute together with an appropriate infectious disease-related antigen asdefined hereafter, will induce and will preferably maintain a state ofimmunotolerance to the antigen, thus enabling to treat an infectiousdisease infection in humans.

In one embodiment, the tolerogenic adjuvant, when combined to aninfectious disease-related antigen induces or maintains immunotoleranceto the viral antigen, thereby treating a related infectious disease.

In one embodiment, non-pathogenic bacteria, especially probiotics andcommensal bacteria, may be used as tolerogenic adjuvant in the contextof the present invention. In a particular embodiment, Lactobacillus sp.,preferably Lactobacillus plantarum and/or Lactobacillus rhamnosus, maybe used as tolerogenic adjuvant in the context of the present invention.

In one embodiment, a combination of non-pathogenic bacteria, such as twoor more commensal bacteria, may be used as the tolerogenic adjuvant inthe context of the present invention.

In another embodiment, instead of or additionally to being attenuated,pathogenic bacteria described herein may be inactivated to be used astolerogenic adjuvant in the context of the present invention, butattenuated pathogenic bacteria may also be used after having beeninactivated.

In some embodiment, the tolerogenic adjuvant, comprising anon-pathogenic bacterium or an attenuated pathogenic bacterium, furthercomprises a prebiotic.

As used herein, “prebiotics” are substances which induce the growth oractivity of certain bacteria. Prebiotics are of different natureincluding e.g. sugars, such as oligosaccharides and polysaccharides.

Any prebiotic may be used in combination with the non-pathogenicbacterium or the attenuated pathogenic bacterium described herein.

In some embodiments, the tolerogenic adjuvant comprises at least oneprebiotic selected from the group consisting of fructooligosaccharides(FOS), galactooligosaccharides (GOS), inulin,trans-galactooligosaccharides (TOS), beneo synergy 1 (SYN1),oligofructose-inulin, lactulose, oat fibera, germinated barley,hydrolyzed guar guma, resistant starcha, plantago ovataa, beta glucanaand pectina.

According to another embodiment of the invention, CD8 vaccine is an exvivo generated dendritic, natural killer or B cell population presentingMHC-II and MHC-1b/E-restricted antigens.

In one embodiment, the ex vivo generated dendritic, natural killer or Bcell population presenting MHC-II and MHC-1b/E-restricted antigens is anactive vaccine.

In one embodiment, the MHC-1b/E-restricted antigen is an infectiousdisease-related pathogen-specific antigen.

In one embodiment, the infectious diseases-related antigen orpathogen-specific antigen is an HIV or SIV-derived MHCIb/E-bindingantigen.

In one embodiment, the HIV-derived MHCIb/E-binding antigen describedherein is selected from the group of SEQ ID NO: 1 to SEQ ID NO: 4.

In one embodiment, the HIV-derived MHCIb/E-binding antigen has an aminoacid sequence selected from the group consisting of the sequenceRMYSPVSIL (SEQ ID NO: 1), the sequence PEIVIYDYM (SEQ ID NO: 2), thesequence TALSEGATP (SEQ ID NO: 3) and the sequence RIRTWKSLV (SEQ ID NO:4).

In some embodiments, the MHC-II-restricted peptide or antigen is anHLA-DR-restricted peptide or antigen. Examples of HLA-DR-restrictedpeptides include for instance the HLA-DR-binding antigens having one ofthe sequences QGQMVHQAISPRTLN (SEQ ID NO: 7) (Gag p24), GEIYKRWIILGLNKI(SEQ ID NO: 8) (Gag p24), KRWIILGLNKIVRMY (SEQ ID NO: 9) (Gag p24) orFRKYTAFTIPSINNE (SEQ ID NO: 10) (Pol RT).

In one embodiment, the HLA-DR-restricted peptides are derived from HIV,preferably from HIV-1.

In one embodiment, the HLA-DR-restricted peptides are HIV-derivedHLA-DR-binding antigen having an amino acid sequence selected from thegroup consisting of the sequence QGQMVHQAISPRTLN (SEQ ID NO: 7) (Gagp24), the sequence GEIYKRWIILGLNKI (SEQ ID NO: 8) (Gag p24), thesequence KRWIILGLNKIVRMY (SEQ ID NO: 9) (Gag p24) and the sequenceFRKYTAFTIPSINNE (SEQ ID NO: 10) (Pol RT).

In one embodiment, dendritic, natural killer or B cell populationpresenting MHC-II and MHC-1b/E-restricted peptides is an allogenic cellpopulation. In a preferred embodiment, dendritic, natural killer or Bcell population presenting MHC-II and MHC-1b/E-restricted restrictedpeptides is an autologous cell population.

As used herein, “allogeneic cells” refers to cells isolated from onesubject (the donor) and infused in another (the recipient or host).

As used herein, “autologous cells” refers to cells that are isolated andinfused back into the same subject (recipient or host).

The present invention thus also relates to an ex vivo method forgenerating dendritic, natural killer or B cell population presentingMHC-II and MHC-1b/E-restricted peptides.

In one embodiment, the ex vivo method for generating dendritic, naturalkiller or B cell population presenting MHC-II and MHC-1b/E-restrictedpeptides, comprises:

-   -   a. optionally, reducing MHC-1a expression in immature dendritic,        natural killer or B cells with an agent inhibiting TAP        expression or activity,    -   b. loading immature dendritic cells with HLA-DR and/or        MHC-1b/E-restricted peptide, and    -   c. maturing the loaded immature dendritic, natural killer or B        cells.

In one embodiment, the immature dendritic cells are produced frommonocytes dendritic cell precursors (MO-DC) precursors.

As used herein, the term “monocytes dendritic cell precursors” refers tomonocytes and other bone marrow precursors (e.g., myeloid precursor).These cells can be isolated from any tissue where they reside,particularly lymphoid tissues such as the spleen, bone marrow, lymphnodes and thymus. Monocytes dendritic cell precursors can be isolatedfrom umbilical cord blood. Monocytes dendritic cell precursors also canbe isolated by any technic well known in the art from peripheral bloodmononuclear cells or bone marrow samples. Monocytes dendritic cellprecursors also can be isolated from frozen samples. Methods forisolating MO-DC precursors and the immature dendritic cells from thevarious sources provided above, including blood and bone marrow, can beaccomplished in a number of ways. Typically, a cell population iscollected from the individual and enriched for the MO-DC precursors. Forexample, a mixed population of cells comprising the MO-DC precursors canbe obtained from peripheral blood by leukapheresis, apheresis, densitycentrifugation, differential lysis, filtration, antibody panning (e.g.,flow cytometry, positive or negative selection) or preparation of abuffy coat. In one embodiment, the MO-DC precursors are non-activatedthus, in one embodiment, the method selected must not activate the MO-DCprecursors. For example, if antibody panning is selected to enrich thecell population for precursors the antibodies selected must not activatethe cells (e.g., through the induction of the influx of calcium ionswhich can result as a consequence of crosslinking the molecules on thesurface to which the antibodies bind). Typically, when antibody panning,antibodies are used that eliminate macrophage, B cells, Natural Killercells, T cells and the like. Antibodies can also be used to positivelyselect for monocyte like cells that express CD14.

In one embodiment, the MO-DC precursors and the immature dendritic cellscan be obtained from autologous PBMCs (peripheral blood mononuclearcells). In one embodiment, the MO-DC precursors and the immaturedendritic cells can be obtained from autologous tissues. In oneembodiment, the MO-DC precursors and the immature dendritic cells can beobtained from an HLA-matched healthy individual.

In one embodiment, the immature dendritic cells can by obtain frominduced pluripotent stem cells (iPS). In one embodiment, the immaturedendritic cells can by obtain from CD34⁺ dendritic cell precursors. Inone embodiment, the immature dendritic cells can by obtain from humandendritic cell lines. In one embodiment, the immature dendritic cellscan by obtain from CD34⁺ dendritic cell precursor cell lines. Anon-limiting example of a cell line that can be used to generateimmature dendritic cell is the CD34⁺ human acute myeloid leukemia cellline (MUTZ-3), see, for example, Masterson et al., (2002) Blood,100:701-703.

In another embodiment, the MO-DC precursors and the immature dendriticcells can be obtained from an HLA-matched healthy individual forconversion to immature dendritic cells, maturation, activation andadministration to an HLA-matched subject in need thereof.

In one embodiment, the cell populations enriched form non-activatedMO-DC precursors or CD34⁺ dendritic cell precursors are cultured ex vivoor in vitro for differentiation, maturation and/or expansion.

Briefly, ex vivo differentiation typically involves culturing the MO-DCprecursors or CD34⁺ dendritic cell precursors, or populations of cellcomprising non-activated MO-DC precursors or CD34⁺ dendritic cellprecursors, in the presence of one or more differentiation agents. Suchagents generally comprise granulocyte-macrophage colony stimulatingfactor (GM-CSF), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin3 (IL-3), stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand(Flt3-L) or a combination thereof. Such agents can be used alone or incombination. For example, the GM-CSF can be used alone or in combinationwith one or more cytokines, such as IL-4, IL-6, IL-3, SC and/or Flt3-L.In one embodiment, the non-activated MO-DC precursors or CD34⁺ dendriticcell precursors are differentiated to form immature dendritic cellscapable of inducing the activation and proliferation of a substantialnumber of T cells.

Suitable culture conditions to product and maintained immature dendriticcell precursors are well known in the art. Such culture media include,without limitation, AIM-V®, RPMI 1640, DMEM, X-VIVO 15®, and the likesupplemented cytokines. The culture media can be supplemented withserum, amino acids, vitamins, divalent cations, and the like, to promotedifferentiation of the cells into dendritic cells. In one embodiment,the dendritic cell precursors can be cultured in a serum-free media.Such culture conditions can optionally exclude any animal-derivedproducts. Typically, GM-CSF is added to the culture medium at aconcentration of about 2 to about 200 ng/ml, or typically 20 ng/ml ofGM-CSF, IL-4 is added to the culture medium at a concentration of about2 to about 200 ng/ml, or typically 20 ng/ml of IL-4. IL-6 is added tothe culture medium at a concentration of about 2 to about 200 ng/ml, ortypically 20 ng/ml of IL-6. IL-3 is added to the culture medium at aconcentration of about 2 to about 200 ng/ml, or typically 20 ng/ml ofIL-3, SCF is added to the culture medium at a concentration of about 10to about 1000 ng/ml, or typically 100 ng/ml of SCF, and Flt3-L is addedto the culture medium at a concentration of about 10 to about 1000ng/ml, or typically 100 ng/ml of Flt3-L. Precursors, when differentiatedto form immature dendritic cells, generally demonstrate a typicalexpression pattern of cell surface proteins seen for immature dendriticcells, e.g., the cells are typically CD14⁻, HLA-DR⁺, CD11c⁺, CD83⁻ andexpress low levels of CD86. A non-limiting example of production ofimmature dendritic cell precursors is described in Example 2. At thisstage, the immature dendritic cells are able to capture soluble antigensvia specialized uptake mechanisms.

In one embodiment, the expression of MHC-1a in immature dendritic cellsor in dendritic cells is reduced by an agent inhibiting TAP expressionor activity.

According to one embodiment, the immature dendritic cells or thedendritic cells express a reduced level of the major histocompatibilityclass 1a (MHC-1a) molecules on their surface. According to oneembodiment, the immature dendritic cells or the dendritic cells do notexpress the major histocompatibility class 1a (MHC-1a) on their surface.

The “MHC class 1a presentation” refers to the “classical” presentationthrough HLA-A, HLA-B and/or HLA-C molecules whereas the MHC class Ibpresentation refers to the “non-classical” antigen presentation throughHLA-E, HLA-F, HLA-G and/or HLA-H molecules.

Methods for inhibiting MHC-1a molecules expression are well-known. Forexample, the inhibition of the TAP transporter (transporter associatedwith antigen processing) leads to a decreased expression of MHC-1amolecules thereby promoting HLA-E molecules expression on the surface ofdendritic cells.

Exemplary methods to inhibit the TAP transporter in the endoplasmicreticulum include, but are not limited to, CRISPR-CAS-9 technology,silencing RNA, transfected DCs with the UL-10 viral protein from the CMV(cytomegalovirus) or the use of viral proteins.

Examples of viral genes or proteins silencing TAP expression include,but are not limited to, HSV-1 ICP47 protein, varicella-virus UL49.5protein, cytomegalovirus US6 protein or gamma herpesvirus EBV BNLF2aprotein, HIV nef protein.

Another method is the use of a chemical product to inhibit theexpression of MHC class 1a molecules without changing HLA-E expressionon the surface of tolerogenic DCs. Examples of chemical productsinclude, but are not limited to, 5′-methyl-5′-thioadenosine orleptomycin B.

In one embodiment, the TAP inhibitor is RNA synthesized from the pGem4Zvector containing the UL49.5 gene from BHV-1.

In one embodiment, the TAP inhibitor can be efficiently introduced intoimmature dendritic cells by electroporation. In another embodiment, theTAP inhibitor can be efficiently introduced into immature dendriticcells by transfection.

Depletion of MHC-1a can be monitored by methods known in the art. Forexample, antibodies can also be used to monitor that immature dendriticcells or dendritic cells are MHC-1a^(−/low).

In one embodiment, the immature dendritic cells or dendritic cells canbe loaded (or pulsed) in the presence of at least one predeterminedantigen. In one embodiment, the expression of MHC-1a in immaturedendritic cells or dendritic cells has been previously reduced. Inanother embodiment, the expression of MHC-1a in immature dendritic cellshas not been previously reduced.

In one embodiment, the immature dendritic cells or dendritic cellspresent peptides or antigens that specifically bind to HLA-DR and/orMHC-1b/E molecules. Thus, in one embodiment, the immature dendritic canbe loaded (or pulsed) by contacting immature dendritic cells ordendritic cells with a predetermined peptide or antigen either prior to,after to or during maturation. In one embodiment, the MHC-1a depletedimmature dendritic cells or dendritic cells present peptides or antigensthat specifically bind to HLA-DR and/or MHC-1b/E molecules. Thus, in oneembodiment, the MHC-1a depleted immature dendritic can be loaded (orpulsed) by contacting immature dendritic cells with a predeterminedpeptide or antigen either prior to, after to or during maturation.

Suitable predetermined antigens for use in the present invention caninclude any infectious-diseases related antigen. Infectious-diseasesrelated antigens are described hereafter and include, for example, HIVor SIV MHC-Ib/E peptides or antigens.

Methods for contacting dendritic cells with antigen are generally knownin the art (See Steel and Nutman, J. Immunol. 160:351-60 (1998); Tao etal., J. Immunol. 158:4237-44 (1997); Dozmorov and Miller, Cell Immunol.178:187-96 (1997); Inaba et al., J Exp Med. 166:182-94 (1987); Macatoniaet al., J Exp Med. 169:1255-64 (1989); De Bruijn et al., Eur. J.Immunol. 22:3013-20 (1992); the disclosures of which are incorporated byreference herein). Typically, the immature dendritic immature cellsobtained by the methods of the present invention can be cultured in thepresence the predetermined antigen under suitable culture conditions, asdescribed above. Optionally, the immature dendritic cells can be admixedwith the predetermined antigen in a typical dendritic cell culture mediawith or without GM-CSF, and/or a maturation agent. Following at leastabout 10 minutes to about 2 days of culture with the antigen, theantigen can be removed and culture media supplemented with a maturationagent. GM-CSF and others cytokines (e.g., such as IL-4) can also beadded to the culture media.

In one embodiment, the immature dendritic cells or dendritic cells canbe transfected with a plasmid coding for MHC-1b/E molecules. In anotherembodiment, the immature dendritic cells or dendritic cells can betransfected with a plasmid coding for peptide-MHC-1b/E complex.

In one embodiment, the immature dendritic cell (optionally MHC-1adepleted and previously loaded) can be matured with a maturation agent.

In on embodiment, the immature dendritic cells can be matured to formmature dendritic cells. Mature dendritic cells lose the ability to takeup antigen and the cells display up-regulated expression ofco-stimulatory cell surface molecules and secrete various cytokines. Forexample, mature dendritic cells can express higher levels of HLA-DRand/or MHC-1b/E antigens and are generally identified as MHC-1a^(/low),CD80⁺, CD83⁺, and CD86⁺. Greater MHC expression leads to an increase inantigen density on the DC surface, while up regulation of co-stimulatorymolecules CD80 and CD86 strengthens the T cell activation signal throughthe counterparts of the co-stimulatory molecules, such as CD28 on the Tcells.

Methods to prepare mature dendritic cells are well known in the art. Forexample, immature dendritic cells can be matured by contacting theimmature dendritic cells with effective amounts or concentrations of adendritic cell maturation agent. Dendritic cell maturation agents caninclude, for example, BCG, IFNγ, LPS, TNFα, IL-1β, IL-6, PGE2, Poly I:C,TLR7/8-ligand, or a combination thereof.

For example, the immature DCs are typically contacted with effectiveamounts of LPS for about one hour to about 48 hours, preferably for 24hours. The immature dendritic cells can be cultured and matured insuitable maturation culture conditions. Suitable tissue culture mediainclude AIM-V®, RPMI 1640, DMEM, X-VIVO 15®, and the like. The tissueculture media can be supplemented with amino acids, vitamins, cytokines,such as GM-CSF, divalent cations, and the like, to promote maturation ofthe cells.

As exemplary purpose, dendritic cells can be matured in presence ofIL-1β, IL-6, PGE2, TNF-α, LPS, Poly I:C. Typically about 2 ng/ml ofIL-1β, 30 ng/ml of IL-6, 1 μg/ml of PGE2, 10 ng/ml of TNF-α, 250 ng/mlof LPS, 150 ng/ml of Poly I:C are generally used.

Maturation of dendritic cells can be monitored by methods known in theart for dendritic cells. Cell surface markers can be detected in assaysfamiliar to the art, such as flow cytometry, immunohistochemistry, andthe like. The cells can also be monitored for cytokine production (e.g.,by ELISA, another immune assay, or by use of an oligonucleotide array).Mature DCs of the present invention also lose the ability to uptakeantigen, which can be analyzed by uptake assays familiar to one ofordinary skill in the art.

Thus, the present invention also relates to a mature dendritic cellpopulation presenting MHC-II and MHC-1b/E-restricted peptides obtainableor obtained by the ex vivo method as described here above.

Instead of dendritic cells, other immune cell types may be used toobtain a mature immune cell population presenting MHC-II andMHC-1b/E-restricted peptides or antigen.

In one embodiment, the CD8 vaccine is an active vaccine which is an exvivo generated natural killer cell population presenting at least oneMHC-1b/E-restricted antigen and at least one MHC-II restricted antigen.

In one embodiment, the natural killer cell is a K562 cell line.

In some embodiment, the natural killer cell is modified to expressMHC-1b/E.

In another embodiment, the CD8 vaccine is an active vaccine which is anex vivo generated natural B population presenting at least oneMHC-1b/E-restricted antigen and at least one MHC-II restricted antigen.

In one embodiment, the B cell is a cell line.

In some embodiment, the B cell is modified to express MHC-1b/E.

Thus, the present invention also relates to a mature natural killer cellpopulation presenting MHC-II and MHC-1b/E-restricted peptides obtainableor obtained by the ex vivo method as described here above.

The present invention also relates to a mature B cell populationpresenting MHC-II and MHC-1b/E-restricted peptides obtainable orobtained by the ex vivo method as described here above.

According to another embodiment of the invention, the CD8 vaccine is anex vivo generated MHC-1b/E-restricted CD8⁺ T cell population.

In one embodiment, ex vivo generated MHC-1b/E-restricted CD8⁺ T cellpopulation is a passive vaccine.

In one embodiment, the MHC-1b/E-restricted CD8⁺ T cell populationrecognizes an MHC-1b/E-restricted infectious diseases-related antigen.

The present invention thus also relates to an ex vivo method forgenerating MHC-1b/E-restricted CD8⁺ T cell population.

In one embodiment, the ex vivo method for generating MHC-1b/E-restrictedCD8⁺ T cell population, comprises:

-   -   a. culturing naïve CD8⁺ T cells in the presence of dendritic,        natural killer or B cell population presenting        MHC-1b/E-restricted peptides enabling generation of        MHC-1b/E-restricted CD8⁺ T cells, and    -   b. expanding the MHC-1b/E-restricted CD8⁺ T cells.

In one embodiment, the CD8⁺ T cells, preferably naïve CD8⁺ T cells, areisolated by any technic well known in the art from a blood sample. Inone embodiment, CD8⁺ T cells, preferably naïve CD8⁺ T cells, areisolated from PBMCs (peripheral blood mononuclear cells) by flowcytometry. In one embodiment, the CD8⁺ T cells, preferably naïve CD8⁺ Tcells, may be isolated from frozen PBMCs. In one embodiment, the CD8⁺ Tcells are allogenic T cells, preferably allogenic naive T cells. Inanother embodiment, the CD8⁺ T cells are autologous T cells, preferablyautologous naive T cells. T cell isolation or purification can beachieved by positive, or negative selection, including but not limitedto, the use of antibodies directed to CD8, CD56, CD57, CD45RO, CD45RA,CCR7, and the like. For example, naive CD8⁺ T cell isolation can beperformed in a one-step or two step procedure. A two-step procedure cancomprise a first step wherein naive T cells are enriched by depletion ofnon-naive T cells, and a second step wherein the enriched naive T cellsare labeled with CD8 antibodies for subsequent positive selection of theCD8⁺ naive T cells.

In one embodiment, the CD8⁺ T cells, preferably naïve CD8⁺ T cells, morepreferably autologous naive CD8⁺ T cells, are stimulated with peptide orantigen pulsed dendritic cells (for example MHC-Ib/E-antigen pulsedtolerogenic dendritic cells) in presence of stimulating agent. Afterstimulation, cells can be washed, for example with PBS, and can bestained with anti-CD8 antibodies and using MHC-peptide pentamer forsorting. The purified CD8⁺ T cells are enriched and may be used for thefollowing activation step.

In one embodiment, the CD8⁺ T cells, preferably naïve CD8⁺ T cells, morepreferably autologous naive CD8⁺ T cells are co-incubated with dendriticcells presenting MHC-1b/E-restricted peptides. In one embodiment, saiddendritic cells presenting MHC-1b/E-restricted peptides also presentMHC-II-restricted peptide.

In one embodiment, the dendritic cells do not express MHC-1a moleculeson their surface. In one embodiment, the dendritic cells express lessthan 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of MHC-1amolecules on their surface (i.e., relative to all MHC moleculesexpressed at the surface of the dendritic cell). In one embodiment, thedendritic cells express at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85,90% or 95% of MHC-1b molecules on their surface. In one embodiment,dendritic cells express only MHC-Ib molecules on their surface.

In one embodiment, dendritic cells express MHC-II molecules on theirsurface. In one embodiment, dendritic cells express MHC-II molecules andMHC-Ib molecules on their surface.

In one embodiment, the CD8⁺ T cells, preferably naïve CD8⁺ T cells, morepreferably autologous naive CD8⁺ T cells, are contacted with thetolerogenic dendritic cells as described hereinabove. Accordingly, atthe end of the culture, the T cells are suppressor MHC-1b/E-restrictedCD8⁺ T cells and can induce immunotolerance.

In one embodiment, the culture for generating the MHC-1b/E-restrictedCD8⁺ T cells of the invention is performed during at least 1 day, atleast 2 days, at least 3 days, at least 4 days, at least 5 days, atleast 6 days, at least 7 days, at least 8 days or more. In oneembodiment, the culture for generating the MHC-1b/E-restricted CD8⁺ Tcells of the invention is performed during at least 1 week, at least 2weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks or more. Inone embodiment, the culture for generating the MHC-1b/E-restricted CD8⁺T cells of the invention is performed during at least 1 month, at least2 months, at least 3 months or more.

In one embodiment, MHC-Ib/E natural killer cells or MHC-Ib/E B cells areused instead of MHC-Ib/E dendritic cells in the method described hereinfor generating an autologous MHC-1b/E-restricted CD8⁺ T cell population.

In one embodiment, the MHC-1b/E-restricted CD8⁺ T cell populationgenerated ex vivo is isolated by flow cytometry based on their abilityto binds to specific HLA-E antigens or peptides (e.g., specifictetramers).

In one embodiment, the isolated MHC-1b/E-restricted CD8⁺ T cellpopulation thus obtained is then expanded ex vivo by culturing thesecells in the presence of at least one T cell activator. Examples of Tcell activator include, but are not limited to, to be completed.Alternatively, other examples of T cell activators that may be usedduring expansion include, but are not limited to, mitogen such asPMA/ionomycin, super-antigen, anti-CD3 antibody and the like.Preferably, the anti-CD3 monoclonal antibody is coated. In oneembodiment, the T cell activator can be used in the presence of feedercells.

Feeder cells include, but are not limited to, ΔCD3 cells (Tcell-depleted accessory cells), irradiated PBMCs, irradiated DCs,artificial APCs (antigen presenting cells), Sf9 cells, insect cells, apool of PBMCs or a pool of B cells from different subjects, KCD40L cellsEBV-transformed B cell lines and EBV-transformed lymphoblastoid cells(LCL).

In another embodiment, the isolated MHC-1b/E-restricted CD8⁺ T cellpopulation thus obtained is then expanded ex vivo by culturing thesecells in the presence of an antigen-specific T cell activator (e.g.,anti-CD3/CD28 antibodies, PMA/iono, cytokines and the likes). In oneembodiment, the antigen-specific T cell activator can be used in thepresence of feeder cells as described here above.

In one embodiment, the culture for expanded the ex vivoMHC-1b/E-restricted CD8⁺ T cells of the invention is performed during atleast 1 day, at least 2 days, at least 3 days, at least 4 days, at least5 days, at least 6 days, at least 7 days, at least 8 days or more. Inone embodiment, the culture for expanded the ex vivo MHC-1b/E-restrictedCD8⁺ T cells of the invention is performed during at least 1 week, atleast 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks ormore. In one embodiment, the culture for expanded the ex vivoMHC-1b/E-restricted CD8⁺ T cells of the invention is performed during atleast 1 month, at least 2 months, at least 3 months or more.

Thus, the present invention also relates to an MHC-1b/E-restricted CD8⁺T cell obtainable or obtained by the ex vivo method as described hereabove.

In one embodiment, the infectious diseases-related antigen is apathogen-specific antigen.

“Pathogenic-specific antigen” can be selected from any organism that isknown to be pathogenic, or against which it is desirable to elicit animmune response. Such pathogen-specific antigens are well known in theart, therefore a suitable antigen can be selected by one of ordinaryskill in the art. The antigen is chosen according to the type ofinfectious disease to be treated. For example, when the disease to beprevented or treated is acquired immune deficiency syndrome (AIDS) or asimian immunodeficiency virus (SIV) infection, the CD8 vaccine comprisesor encodes an antigen from HIV or SIV respectively.

In one embodiment, the pathogen-specific antigen described herein can bederived from any human or animal pathogen. In one embodiment, thepathogen specific antigen is a viral pathogen, a bacterial pathogen, ora parasite, and the antigen may be a protein derived from the viralpathogen, bacterial pathogen, or parasite. In one embodiment, theparasite may be an organism or disease caused by an organism. Forexample, the parasite may be a protozoan organism, a protozoan organismcausing a disease, a helminth organism or worm, a disease caused by ahelminth organism, an ectoparasite, or a disease caused by anectoparasite.

In one embodiment, the pathogen-specific antigen described herein can bean antigen from a viral pathogen. In one embodiment, thepathogen-specific antigen can be an antigen from a bacterial pathogen.In one embodiment, the pathogen-specific antigen can be an antigen froma parasitic organism. In another embodiment, the pathogen-specificantigen can be an antigen from a helminth organism.

In one embodiment, the pathogen-specific antigen described herein isnon-infectious.

In one embodiment, when recombinant virus or bacteria are used toexpress the antigen, these are preferably inactivated microorganisms.

In one embodiment, the pathogen-specific antigen described herein is aparticulate antigen.

In one embodiment, the pathogen-specific antigen described herein mayresult from the expression of a viral nucleic acid sequenceadvantageously contained into an appropriate recombinant microorganism.In one embodiment said recombinant microorganism is a CMV, preferably aCMV vector as described herein above. In another embodiment, saidrecombinant microorganism is a bacterium, preferably a differentbacterium from the non-pathogenic bacterium as described herein above.

In one embodiment, the pathogen-specific antigen described herein can becodon optimized. Many viruses, including HIV and other lentiviruses, usea large number of rare codons and, by altering these codons tocorrespond to codons commonly used in the desired subject (for example,humans), enhanced expression of the antigens can be achieved. Forexample, rare codons used in HIV proteins can be mutated into those thatappear frequently in highly expressed human genes (Andre et al. (1998) JVirol 72, 1497-1503).

In one embodiment, the pathogen-specific antigen described herein can beconsensus sequences or mosaic antigens containing sequence fragmentsfrom different strains of pathogens.

In one embodiment, the particulate antigen described herein is a viralantigen.

In one embodiment, the particulate antigen is selected from viralparticles, recombinant viral particles, virus-like particles,recombinant viral particles, polymeric microparticles presenting ontheir surface one or more viral peptides or epitopes, conjugate viralproteins and concatemer viral proteins.

In one embodiment, the particulate antigen described herein may be oneor more viral proteins or peptides, recombinant or not, either in theform of conjugates or of concatemers.

In one embodiment, the pathogen-specific antigen described herein can bederived from the human immunodeficiency virus (HIV), simianimmunodeficiency virus (SIV), herpes simplex virus, hepatitis B virus,hepatitis C virus, papillomavirus, Plasmodium parasites, andMycobacterium tuberculosis.

In one embodiment, the pathogen-specific antigen described herein is anHIV or SIV antigen. In one embodiment, the HIV or SIV antigen isselected from the group consisting of any HIV or SIV strains. In oneembodiment, the HIV or SIV antigen is selected from the group consistingof gag, tat, pol, vif, nef and env antigens.

In one embodiment, the pathogen-specific antigen described herein isderived from immunogenic apoptotic bodies from infected cells or derivedfrom tissue lysate.

Infected cells may derive from tissue biopsy or from expansion ofcirculatory infected cells. Said infected cells can be infected byvirus, bacteria, parasitic organisms or helminth organisms.

Immunogenic apoptotic bodies from infected cells may be obtained forexample with anthracyclines including doxorubicin, daunorubicin,idarubicin and mitoxanthrone; oxaliplatin, UVC, UVB or y-radiationtreated infected cells releasing apoptotic bodies.

Examples of tissue lysate include, but are not limited to, lymph nodes,synovial liquid or inflammatory tissue lysate.

In one embodiment, the pathogen-specific antigen described herein is anantigen from HIV or SIV origin.

In one embodiment, the immunogenic bodies are obtained from HIV infectedCD4⁺ T cells.

In one embodiment, the pathogen-specific antigen described herein is anantigen from HIV origin.

In one embodiment, the pathogen-specific antigen described herein is anHIV antigen.

Due to the great variability in the HIV genome, which results frommutation, recombination, insertion and/or deletion, HIV has beenclassified in groups, subgroups, types, subtypes and genotypes. Thereare two major HIV groups (HIV-1 and HIV-2) and many subgroups becausethe HIV genome mutates constantly. The major difference between thegroups and subgroups is associated with the viral envelope. HIV-1 isclassified into a main group (M), said group M being divided into leastnine genetically distinct subtypes. These are subtypes A, B, C, D, F, G,H, J and K. Many other subtypes resulting from in vivo recombination ofthe previous ones also exist (e.g., CRF). In one embodiment, the HIVantigen is related to a specific HIV group, subgroup, type, subtype orto a combination of several subtypes.

In one embodiment, the HIV virus is HIV-1 or HIV-2, preferably, HIV-1.In another embodiment, the HIV-1 virus is from group M and subtype B(HXB2).

In one embodiment, the HIV antigen is an inactivated whole HIV virus.

As used herein, “inactivated whole HIV” means a complete HIV particle,which has been inactivated, and which is no more infectious.

In one embodiment, the HIV antigen is an autologous HIV antigen. Inanother embodiment, the HIV antigen is not an autologous HIV antigen. Inone embodiment, the HIV antigen is made of inactivated autologous HIVvirus.

As used herein, “an antigen made of inactivated autologous HIV virus”refers to an antigen comprising or consisting of the HIV virus infectingthe human to be treated and appropriately inactivated for a safetherapeutic administration to humans. Thus, in practice, to prepare thevaccine composition of the invention, the HIV virus is isolated from thehuman to be treated, more particularly from the CD4⁺ T cells of saidhuman. The thus isolated HIV virus is cultured and inactivated.

In one embodiment, the HIV antigen is selected from the group consistingof a HIV gag, a HIV env, a HIV rev, a HIV tat, a HIV nef, a HIV pol, anda HIV vif.

In one embodiment, the HIV antigen comprises one or more epitopes of aHIV gag, a HIV env, a HIV rev, a HIV tat, a HIV nef, a HIV pol, and aHIV vif proteins.

In one embodiment, the HIV antigen comprises at least a HIV gag and/orHIV pol protein. Alternatively or additionally, said antigen derivedfrom a HIV virus may comprise one or more proteins encoded by gag suchas the capsid protein (p24) and the matrix protein (p1), and/or one ormore proteins encoded by pol such as the integrase, the reversetranscriptase and the protease.

In one embodiment, the pathogen-specific antigen described herein is aMHCIb/E-binding antigen. In one embodiment, the pathogen-specificantigen is a MHCIb/E-binding peptide.

In one embodiment, the pathogen-specific antigen described herein is anHIV or SIV-derived MHCIb/E-binding peptide. In one embodiment, thepathogen-specific antigen is an HIV or SIV-derived MHCIb/E-bindingantigen.

In one embodiment, the HIV-derived MHCIb/E-binding antigen describedherein is selected from the group of SEQ ID NO: 1 to SEQ ID NO: 4. Inone embodiment, the HIV-derived MHCIb/E-binding peptide is selected fromthe group of SEQ ID NO: 1 to SEQ ID NO: 4.

In one embodiment, the HIV-derived MHCIb/E-binding antigen has the aminoacid sequence RMYSPVSIL (SEQ ID NO: 1). In one embodiment, theHIV-derived MHCIb/E-binding antigen has the amino acid sequencePEIVIYDYM (SEQ ID NO: 2). In one embodiment, the HIV-derivedMHCIb/E-binding antigen has the amino acid sequence TALSEGATP (SEQ IDNO: 3). In one embodiment, the HIV-derived MHCIb/E-binding antigen hasthe amino acid sequence RIRTWKSLV (SEQ ID NO: 4).

As used herein, the term “alpha interferon” (IFN-α) or“interferon-alpha” refers to a family of more than 20 related butdistinct members encoded by a cluster on chromosome 9 and all bind tothe same IFN receptor. Among these, the IFN-α2 have 3 recombinantvariants (α2a, α2b, α2c) depending upon the cells of origin and theIFN-α2b is the predominant variant in human genome. There is evidencethough that each subtype has a different binding capacity to the IFNAR,modulating the signaling transduction events and the biological effectsin the target cells.

In one embodiment, the interferon-alpha blocking agent described hereinis an agent neutralizing circulating IFN-α and/or an agent blockingIFN-α signaling, and/or an agent depleting IFN-α producing cells, and/oran agent blocking IFN-α production.

In one embodiment, the interferon-alpha blocking agent described hereincomprises at least one agent selected from: an agent neutralizingcirculating IFN-α and/or an agent blocking IFN-α signaling, and/or anagent depleting IFN-α producing cells, and/or an agent blocking IFN-αproduction.

In one embodiment, the agent neutralizing circulating IFN-α and/or theagent blocking IFN-α signaling, and/or the agent depleting IFN-αproducing cells, and/or the agent blocking IFN-α production is/are anIFN-α antagonist.

In some embodiments, wherein the interferon-alpha blocking agent isselected from the group consisting of: an agent neutralizing circulatingalpha interferon, an agent blocking interferon-alpha signaling, an agentdepleting IFN-α producing cells, and/or an agent blocking IFN-αproduction, wherein the agent neutralizing circulating alpha interferonis selected from the group comprising active anti-IFN-α vaccineincluding antiferon or passive anti-IFN-α vaccine including anti-IFN-αantibodies or anti-IFN-α hyper-immune serum, wherein the blocking agentof interferon-alpha signaling is selected from the group of anti-type Iinterferon R1 or R2 antibodies or from interferon-alpha endogenousregulators including SOSC1 or aryl hydrocarbon receptors, wherein theagent depleting IFN-α producing cells is an agent depleting plasmacytoiddendritic cells (pDCs), and wherein the agent blocking IFN-α productionis an agent blocking the production of IFN-α by pDCs.

In some embodiments, the interferon-alpha blocking agent is an agentneutralizing circulating alpha interferon selected from the groupconsisting of active anti-IFN-α vaccine including antiferon or passiveanti-IFN-α vaccine including anti-IFN-α antibodies or anti-IFN-αhyper-immune serum, and wherein the blocking agent of interferon-alphasignaling is selected from the group consisting of anti-type Iinterferon R1 or R2 antibodies, SOSC1 and aryl hydrocarbon receptors.

As used herein, the term “alpha interferon antagonist” refers to asubstance which interferes with or inhibits the IFN-α biologicalactivity. “IFN-α biological activity” as used herein refers to anyactivity occurring as a result of IFN-α binding to its receptor IFNAR(IFNAR1/IFNAR2 heterodimer). Such binding can, for example, activate theJAK-STAT signaling cascade, and trigger tyrosine phosphorylation of anumber of proteins including JAKs, TYK2, STAT proteins. Thus, thesignaling blocking agent of interferon can neutralize the fixation ofthe INF-α to its receptor and/or block the signaling cascade induced bythe binding of IFN-α to its receptor. In some embodiments, the IFN-αantagonist is selected from the group of active anti-IFN-α vaccine(e.g., antiferon) or passive anti-IFN-α vaccine (e.g., anti-IFN-αantibody or anti-IFN-α hyper-immune serum). See for example Noel et al.(2018). Cytokine Growth Factor Rev 40:99-112.

In one embodiment, the agent neutralizing circulating IFN-α describedherein is an IFN-α ligand inhibitor.

In one embodiment, the agent neutralizing circulating IFN-α is ananti-IFN-α antibody. Examples of anti-IFN-α antibodies include, withoutlimitation, Sifalimumab, Rontalizumab, MMHA-1 clone, MMHA-2 clone,MMHA-6 clone, MMHA-8 clone, MMHA-9 clone, MMHA-11 clone, MMHA-13 cloneand MMHA-17 clone.

In one embodiment, the agent neutralizing circulating IFN-α is ananti-IFN-α hyper-immune serum.

In one embodiment, the agent neutralizing circulating IFN-α describedherein is an antiferon, such as, for example, an IFN-α-Kinoid.

In one embodiment, the agent neutralizing circulating IFN-α is a solublereceptor that binds IFN-α.

In one embodiment, the agent neutralizing circulating IFN-α does notneutralize circulating type III interferon.

In one embodiment, the agent blocking IFN-α signaling described hereinis an IFNAR antagonist.

In one embodiment, the agent blocking IFN-α signaling is an IFNAR1antagonist. In another embodiment, the agent blocking IFN-α signaling isan IFNAR2 antagonist.

In one embodiment, the agent blocking IFN-α signaling is an antibodythat binds to IFNAR1 or IFNAR2.

In one embodiment, the agent blocking IFN-α signaling is an agent thatantagonizes the type I IFN signaling pathway.

In one embodiment, the agent blocking the agent blocking IFN-α signalingcan be an inhibitor of type I IFN signaling pathway. Type I IFNsignaling pathway inhibitors are well known in the art and include,without limitation, JAK1/2 inhibitors and STAT inhibitors. Accordingly,in one embodiment, the agent blocking IFN-α signaling is selected fromJAK1/2 inhibitors and STAT inhibitors. Non-limiting examples of JAK1/2inhibitor comprise Ruxolitinib, Tofacitinib and Baricitinib.

In one embodiment, the agent blocking IFN-α signaling can be anendogenous negative regulator of type I IFN signaling pathway.Endogenous negative regulators are well known in the art and include,without limitation, SOCS1/3, FOXO3, Aryl hydrocarbon Receptor (AhR) orother negative regulators. Accordingly, in one embodiment, the agentblocking interferon signaling is selected from SOCS1/3, FOXO3 or Arylhydrocarbon Receptor (AhR).

In one embodiment, the agent blocking IFN-α signaling is a PASylatedantagonist. PASylated antagonist of type I IFN are known in the art, seefor example Nganou-Makamdop et al. (2018). PLoS Pathog 14(8): e1007246.

In one embodiment, the IFN-α antagonist described herein is an agentdepleting IFN-α producing cells.

As used herein, the term “IFN-α producing cells” refers to any cell thatproduce IFN-α. In particular, it is well known in the art that theplasmacytoid dendritic cells (pDCs) are the main producer of IFN-α.Thus, in one embodiment, the agent depleting IFN-α producing cellsdepletes pDCs.

In one embodiment, the agent depleting IFN-α producing cells is anantibody. In one embodiment, the antibody depletes pDCs, such as, forexample, an anti-CD123 antibody (i.e., anti-IL-3RA).

In one embodiment, the IFN-α antagonist described herein is an agentthat blocks the production of IFN-α.

In one embodiment, the agent that blocks the production of the IFN-α isan antibody.

In one embodiment, the antibody blocks the production of IFN-α by pDCs.Said antibody can be, for example, an anti-BDCA2 (Blood DC Antigen 2)antibody.

In one embodiment, the agent blocking interferon signaling does notblock type III interferon signaling.

In some embodiments, the interferon-alpha blocking agent selected fromthe group consisting of:

-   -   an anti-IFN-α antibody, preferably Sifalimumab, Rontalizumab,        MMHA-1 clone, MMHA-2 clone, MMHA-6 clone, MMHA-8 clone, MMHA-9        clone, MMHA-11 clone, MMHA-13 clone or MMHA-17 clone,    -   an anti-IFN-α hyper-immune serum,    -   an antiferon, preferably an IFN-α-Kinoid,    -   a soluble receptor that binds IFN-α,    -   an IFNAR1 or IFNAR2 antagonist, preferably an antibody that        binds to IFNAR1 or IFNAR2,    -   a type I IFN signaling pathway inhibitors selected from a STAT        inhibitor and a JAK1/2 inhibitor, such as e.g. Ruxolitinib,        Tofacitinib or Baricitinib,    -   an endogenous negative regulator of type I IFN signaling pathway        selected from SOCS1/3, FOXO3, Aryl hydrocarbon Receptor (AhR) or        another negative regulator,    -   a PASylated antagonist,    -   an antibody depleting pDCs, preferably an anti-CD123 (i.e.        anti-IL-3RA) antibody,    -   an antibody blocking the production of IFN-α by pDCs, preferably        an anti-BDCA2 (Blood DC Antigen 2) antibody.

As used herein, the term “type III interferon”, also calledinterferon-lambda (IFN-k), refers to naturally occurring and/orrecombinant cytokines of the type III interferon-lambda family There arefour IFN-λ members in humans, IFN-λ1/IL-29, IFN-λ2/IL-28A,IFN-λ3/IL-28B,

In one embodiment, the type III interferon is IFN-λ.

In one embodiment, the IFN-λ comprises at least one IFN-λ subtype (e.g.,IFN-λ1, IFN-λ2 IFN-λ4).

In one embodiment, the IFN-λ is selected from the group of IFN-λ1,IFN-λ2 IFN-λ3, IFN-λ4 or a combination thereof.

In one embodiment, the human IFN-21 has the following accession numberNP_742152.1. In one embodiment, the human IFN-λ2 has the followingaccession number NP_742150.1. In one embodiment, the human IFN-λ3 hasthe following accession numbers NP_001333866.1 (isoform 1) orNP_742151.2 (isoform 2). In one embodiment, the human IFN-24 has thefollowing accession number NP_001263183.2.

In one embodiment, the interferon-lambda is IFN-2.1. In one embodiment,the interferon-lambda is IFN-λ2. In one embodiment, theinterferon-lambda is IFN-λ3. In one embodiment, the interferon-lambda isIFN-λ4.

In one embodiment, the interferon-lambda is chemically modified toimprove certain properties such as serum half-life. In one embodimentthe interferon-lambda of the invention is pegylated (i.e., theinterferon-lambda is covalently attached to poly(ethyleneglycol), andthe like). Methods for producing pegylated proteins are well known inthe art, see for example Chapman A et al., 2002, Advanced Drug DeliveryReviews 54: 531-545.

In one embodiment, the interferon-lambda is a functional mimetic ofIFN-λ1. In one embodiment, the interferon-lambda is a functional mimeticof IFN-λ2. In one embodiment, the interferon-lambda is a functionalmimetic of IFN-λ3. In one embodiment, the interferon-lambda is afunctional mimetic of IFN-λ4.

As used herein, the term “functional mimetic” means a molecule which hasthe same or similar biological effects as the naturally occurringprotein. For example an interferon-lambda functional mimetic mayactivate the interferon-lambda receptor and drive the transcription ofIFN-stimulated genes.

In one embodiment, the interferon-lambda functional mimetic is afragment of IFN-λ1. In one embodiment, the interferon-lambda functionalmimetic is a fragment of IFN-λ2. In one embodiment, theinterferon-lambda functional mimetic is a fragment of IFN-λ3. In oneembodiment, the interferon-lambda functional mimetic is a fragment ofIFN-λ4.

In one embodiment, the interferon-lambda functional mimetic is anantibody. Such interferon-lambda functional mimetic antibody can elicitthe same or similar biological effects as the naturally occurringprotein. For example the antibody may bind to an epitope on theinterferon-lambda receptor, activating receptor signaling and drivingthe transcription of IFN-stimulated genes. The heterodimeric receptorcomplex of interferon-lambda (IFNLR) comprises IFNLR1 (IFNLRA, IL-28RA),and IL10R2 (IL-10RB). IFNLR1 confers ligand specificity and enablesreceptor assembly, while IL10R2 is shared with IL-10 family members andis required for signaling.

In one embodiment, the interferon-lambda derivative is a small moleculechemical entity (such as, for example, a chemical entity with amolecular weight less than 900 Daltons). Methods of screening chemicallibraries to identify small molecule chemical entities which may bepotential drug candidates are known in the art. For example, a chemicallibrary may be tested in a ligand-receptor binding assay.

In one embodiment, the agent stimulating the production of type IIIinterferon is an agent that stimulates pattern-recognition receptors(PRRs).

In one embodiment, the agent stimulating the production of type IIIinterferon is an agent that stimulates pattern-recognition receptors(PRRs).

“Pattern-recognition receptor” includes mainly Toll-like receptors(TLRs), NOD-like receptors (NLRs), RIG-1-like receptors (RLRs), andC-type lectin receptors (CLRs). They recognize different microbialsignatures or host-derived danger signals and trigger an immuneresponse, such as interferon production.

In one embodiment, the agent stimulating the production of type IIIinterferon comprises toll-like receptor (TLR) ligands (e.g., TLR3, TLR5,TLR7/8 and TLR9), RIG-I ligands and MDA-5 ligands.

In one embodiment, the agent stimulating the production of type IIIinterferon comprises poly I:C, CpG and/or Tat protein.

In one embodiment, the agent stimulating the production of type IIIinterferon can also induces production of type I interferon.

The present invention further relates to a combination comprising:

-   -   1) a first part comprising CD8 vaccine specific for at least one        infectious disease-related antigen as described herein,    -   2) optionally a second part comprising an interferon-alpha        blocking agent as described herein, and    -   3) a third part comprising a type III interferon and/or an agent        stimulating the production of type III interferon as described        herein.

The present invention also relates to a kit-of-parts comprising at least2 parts:

-   -   1) a first part comprising CD8 vaccine specific for at least one        infectious disease-related antigen as described herein,    -   2) optionally a second part comprising an interferon-alpha        blocking agent as described herein, and    -   3) a third part comprising a type III interferon and/or an agent        stimulating the production of type III interferon as described        herein.

In one embodiment, the CD8 vaccine specific for at least one infectiousdisease-related antigen is comprised in a composition.

In one embodiment, said composition consists essentially of the CD8vaccine specific for at least one infectious disease-related antigen.

As used herein, “consisting essentially of”, with reference to acomposition, means that the CD8 vaccine is the only one therapeuticagent or agent with a biologic activity within said composition.

In one embodiment, said composition is a pharmaceutical composition andfurther comprises at least one pharmaceutically acceptable excipient.

As used herein, the term “excipient” refers to any and all conventionalsolvents, dispersion media, fillers, solid carriers, aqueous solutions,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. In general, the nature of the excipientwill depend on the particular mode of administration being employed. Forinstance, parenteral formulations usually comprise injectable fluidsthat include pharmaceutically and physiologically acceptable fluids suchas water, physiological saline, balanced salt solutions, aqueousdextrose, glycerol or the like as a vehicle. For solid compositions(such as powder, pill, tablet, or capsule forms), conventional non-toxicsolid carriers can include, for example, pharmaceutical grades ofmannitol, lactose, starch, or magnesium stearate. In addition tobiologically neutral carriers, pharmaceutical compositions to beadministered can contain minor amounts of non-toxic auxiliarysubstances, such as wetting or emulsifying agents, preservatives, and pHbuffering agents and the like, for example sodium acetate or sorbitanmonolaurate.

For human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required byregulatory offices, such as, for example, FDA Office or EMA. In oneembodiment, the excipient is an adjuvant, a stabilizer, an emulsifier, athickener, a preservative, an antibiotic, an organic or inorganic acidor its salt, a sugar, an alcohol, an antioxidant, a diluent, a solvent,a filler, a binder, a sorbent, a buffering agent, a chelating agent, alubricant, a coloring agent, or any other component

By “pharmaceutically acceptable” is meant that the ingredients of apharmaceutical composition are compatible with each other and notdeleterious to the subject to which it is administered. Examples ofpharmaceutically acceptable excipient include, but are not limited to,water, saline, phosphate buffered saline, dextrose, glycerol, ethanoland the like or combinations thereof.

Pharmaceutically acceptable excipients that may be used in thepharmaceutical combination of the invention include, but are not limitedto, ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances (for example sodium carboxymethylcellulose), polyethyleneglycol, polyacrylates, waxes, polyethylene-polyoxypropylene-blockpolymers, polyethylene glycol and wool fat.

In one embodiment, said composition is a vaccine composition. In oneembodiment, said vaccine composition further comprises at least oneadjuvant.

In one embodiment, the CD8 vaccine specific for at least one infectiousdisease-related antigen is comprised in a medicament.

In one embodiment, when CD8 vaccine according to the invention comprisesan infectious disease-related antigen and a non-pathogenic bacterium,the infectious disease-related antigen and the non-pathogenic bacteriumare two separate and distinct components that are contained as a mixtureinto a pharmaceutical composition. In another embodiment, when CD8vaccine according to the invention comprises an infectiousdisease-related antigen and a non-pathogenic bacterium, the infectiousdisease-related antigen and the non-pathogenic bacterium, are the samecomponent that are contained into a pharmaceutical composition.

In one embodiment, the CD8 vaccine is a composition, a pharmaceuticalcomposition or a medicament, wherein the CD8 vaccine is conjugated to adelivery vehicle.

In one embodiment, the CD8 vaccine comprises at least one infectiousdisease-related antigen and a non-pathogenic bacterium, wherein the atleast one infectious disease-related antigen and/or the non-pathogenicbacterium is/are conjugated to a delivery vehicle. In one embodiment,the at least one infectious disease-related antigen is conjugated to adelivery vehicle. In one embodiment, the non-pathogenic bacterium isconjugated to a delivery vehicle. In one embodiment, the at least oneinfectious disease-related antigen and non-pathogenic bacterium areconjugated to a delivery vehicle.

By the term “conjugated” is meant that the infectious disease-relatedantigen and/or non-pathogenic bacterium is/are physically or chemicallycoupled, adhered, absorbed or encapsuled to a delivery vehicle. Examplesof conjugation include, without limitation, covalent linkage andelectrostatic complexation. The terms “complexed,” “complexed with,” and“conjugated” are used interchangeably herein. In one embodiment, morethan one copy or type of infectious disease-related antigen isconjugated to a delivery vehicle. In one embodiment, more than one copyor type of non-pathogenic bacterium is conjugated to a delivery vehicle.

Delivery vehicles are well known in the art. For example, the deliveryvehicle can be chosen from a cationic lipid, a liposome, a cochleate, avirosome, an immune-stimulating complex (ISCOM®), a microparticle, amicrosphere, a nanosphere, a unilamellar vesicle (LUV), a multilamellarvesicle, an emulsome, and a polycationic peptide, a lipoplexe, apolyplexe, a lipopolyplexe, a water-in-oil (W/O) emulsion, anoil-in-water (O/W) emulsion, a water-in-oil-in water (W/O/W) multipleemulsion, a micro-emulsion, a nano-emulsion, a micelle, a dendrimer, avirosome, a virus-like particle, a polymeric nanoparticle (such as ananobead, a nanosphere or a nanocapsule), a polymeric microparticle(such as a microsphere or a microcapsule), a chitosan, a poly(lacticacid) (PLA) polymer, a poly(lactic-co-glycolide) (PLGA) polymer, acyclodextrin, a niosome, or an ISCOM® and, optionally, apharmaceutically acceptable carrier. In one embodiment, the deliveryvehicle is in an adapted form for an oral administration, an injection,a topical administration or a rectal administration.

In one embodiment, the CD8 vaccine is conjugated to a nanoparticle, suchas e.g. a nanobead, a nanosphere or a nanocapsule. Preferably, thenanoparticle has a diameter of between 50 and 300 nm, more preferably ofbetween 70 and 200 nm, even more preferably of between 100 and 150 nm.

Microfold cells (or M cells) are found in the gut-associated lymphoidtissue (GALT) of the Peyer's patches in the small intestine, and in themucosa-associated lymphoid tissue (MALT) of other parts of thegastrointestinal tract. These cells are known to initiate mucosalimmunity responses.

In some embodiments, the delivery vehicle is coated with, or conjugatedto, molecules, such as e.g. lectins or peptides, that enhance deliveryto M cells.

M cells express a specific carbohydrate moiety (a-L-fucose) on theapical surface. Lectin subtypes, such as Ulex europaeus agglutinin 1(UEA-1) and Aleuria aurantia, have shown their high specificity forα-L-fucose on M cells. Thus, in some embodiments, the delivery vehicleis coated with, or conjugated to, at least one lectin chosen from Ulexeuropaeus agglutinin 1 (UEA-1) and Aleuria aurantia.

M cells also express claudin 4 and TM4SF3. Delivery system usingsurface-conjugated peptides having high affinity to claudin 4, such ase.g. CTGKSC (SEQ ID NO: 11), LRVG (SEQ ID NO: 12), or CKSTHPLSC (CKS9)(SEQ ID NO: 13), may also be used. Thus, in some embodiments, thedelivery vehicle is coated with, or conjugated to, at least one peptidechosen from CTGKSC (SEQ ID NO: 11), LRVG (SEQ ID NO: 12), and CKSTHPLSC(CKS9) (SEQ ID NO: 13).

In one embodiment, the interferon-alpha blocking agent is comprised in acomposition. In one embodiment, said composition comprises at least oneinterferon-alpha blocking agent selected among: an agent neutralizingcirculating alpha interferon, and/or an agent blocking interferon-alphasignaling, and/or an agent depleting IFN-α producing cells, and/or anagent blocking IFN-a production.

In one embodiment, said composition consists essentially of the agentneutralizing circulating alpha interferon. In one embodiment, saidcomposition consists essentially of the agent blocking IFN-α signaling.In one embodiment, said composition consists essentially of the agentdepleting IFN-α producing cells. In one embodiment, said compositionconsists essentially of the agent blocking IFN-α production.

In one embodiment, said composition is a pharmaceutical composition andfurther comprises at least one pharmaceutically acceptable excipient.

In one embodiment, the interferon-alpha blocking agent is comprised in amedicament.

In one embodiment, said medicament comprises at least oneinterferon-alpha blocking agent selected among: an agent neutralizingcirculating alpha interferon, and/or an agent blocking interferon-alphasignaling, and/or an agent depleting IFN-α producing cells, and/or anagent blocking IFN-α production.

In one embodiment, the agent neutralizing circulating alpha interferonis comprised in a medicament. In one embodiment, the agent blockinginterferon-alpha signaling is comprised in a medicament. In oneembodiment, the agent depleting IFN-α producing cells is comprised in amedicament. In one embodiment, the agent blocking IFN-α production iscomprised in a medicament.

In one embodiment, the type III interferon and/or the agent stimulatingthe production of type III interferon is/are comprised in a composition.

In one embodiment, said composition consists essentially of the type IIIinterferon. In one embodiment, said composition consists essentially ofthe agent stimulating the production of type III interferon. In oneembodiment, said composition consists essentially of the type IIIinterferon and the agent stimulating the production of type IIIinterferon.

In one embodiment, said composition is a pharmaceutical composition andfurther comprises at least one pharmaceutically acceptable excipient.

In one embodiment, the type III interferon and/or the agent stimulatingthe production of type III interferon is/are comprised in a medicament.

In one embodiment, the type III interferon is comprised in a medicament.In one embodiment, the agent stimulating the production of type IIIinterferon is comprised in a medicament. In one embodiment, the type IIIinterferon and the agent stimulating the production of type IIIinterferon are comprised in a medicament.

Another object of the invention is a pharmaceutical compositioncomprising a combination of 1) a CD8 vaccine specific for at least oneinfectious disease-related antigen as described hereinabove, 2)optionally an interferon-alpha blocking agent, and 3) a type IIIinterferon and/or an agent stimulating the production of type IIIinterferon, and further comprises at least one pharmaceuticallyacceptable excipient, or a kit-of-parts as described hereinabove, foruse in the treatment of an infectious diseases in a subject in needthereof.

Another object of the invention is a medicament comprising a combinationof 1) a CD8 vaccine specific for at least one infectious disease-relatedantigen as described hereinabove, 2) optionally an interferon-alphablocking agent, and 3) a type III interferon and/or an agent stimulatingthe production of type III interferon, or a pharmaceutical combinationas described hereinabove, or a kit-of parts as described hereinabove,for use in the treatment of an infectious diseases in a subject in needthereof.

As mentioned hereinabove, the combination, pharmaceutical combination,medicament or kit-of-parts according to the invention of the inventionare to be administered either simultaneously, separately or sequentiallywith respect to each other.

In one embodiment, according to the present invention, 1) the CD8vaccine specific for at least one infectious disease-related antigen, 2)the interferon-alpha blocking agent, and/or 3) the type III interferonand/or an agent stimulating the production of type III interferon in acombination of the invention are to be administered eithersimultaneously, separately or sequentially with respect to each other.

According to one embodiment, 1) the CD8 vaccine specific for at leastone infectious disease-related antigen, 2) interferon-alpha blockingagent, and/or 3) the type III interferon and/or an agent stimulating theproduction of type III interferon, the combination or pharmaceuticalcombination thereof, medicament or kit-of-parts according to theinvention will be formulated for administration to the subject.

In one embodiment, 1) the CD8 vaccine specific for at least oneinfectious disease-related antigen, 2) interferon-alpha blocking agent,and/or 3) the type III interferon and/or an agent stimulating theproduction of type III interferon, the combination or pharmaceuticalcombination thereof, or medicament according to the invention may beadministered orally, intragastrically, parenterally, topically, byinhalation spray, rectally, nasally, buccally, preputiallly, vaginallyor via an implanted reservoir.

In one embodiment, the oral administration comprises mucosaladministration. A “mucosal administration” is a delivery to a mucosalsurface, such as sub-lingual, tracheal, bronchial, pharyngeal,esophageal, gastric, and mucosae of the duodenum, small and largeintestines, including the rectum mucosae. Yet preferably, the mucosalsurface refers to digestive mucosa.

In one embodiment, the administration of each part of the combination,pharmaceutical combination, medicament or kit-of-parts according to theinvention can be done by the same route of administration or by adifferent route of administration.

In one embodiment, the combination, pharmaceutical combination,medicament or kit-of-parts according to the invention is in an adaptedform for an oral or an intragastric administration. Thus, in oneembodiment, the combination, pharmaceutical combination, medicament orkit-of-parts according to the invention is to be administered orally orintragastrically to the subject, for example as a powder, a tablet, acapsule, and the like or as a tablet formulated for extended orsustained release.

Examples of forms adapted for oral or intragastric administrationinclude, without being limited to, liquid, paste or solid compositions,and more particularly tablets, tablets formulated for extended orsustained release, capsules, pills, dragees, liquids, gels, syrups,slurries, suspensions, and the like.

In one embodiment, the CD8 vaccine specific for at least one infectiousdisease-related antigen as described hereinabove is in an adapted formfor an oral or intragastric administration. Thus, in one embodiment, theCD8 vaccine specific for at least one infectious disease-related antigenas described hereinabove is to be administered orally orintragastrically to the subject, for example as a capsule or as atablet.

In another embodiment, the interferon-alpha blocking agent as describedhereinabove is in an adapted form for an oral or intragastricadministration. Thus, in one embodiment, the interferon-alpha blockingagent interferon-alpha as described hereinabove is to be administeredorally or intragastrically to the subject, for example as a capsule oras a tablet.

In another embodiment, the type III interferon and/or the agentstimulating the production of type III interferon as describedhereinabove is/are in an adapted form for an oral or intragastricadministration. Thus, in one embodiment, the type III interferon and/orthe agent stimulating the production of type III interferon as describedhereinabove is/are to be administered orally or intragastrically to thesubject, for example as a capsule or as a tablet.

In one embodiment, the combination, pharmaceutical combination,medicament or kit-of-parts according to the invention is in a formadapted for parenteral administration.

In another embodiment, the combination, pharmaceutical combination,medicament or kit-of-parts according to the invention is in an adaptedform for an injection such as, for example, for intravenous,subcutaneous, intramuscular, intraperitoneal intradermal, transdermalinjection or infusion. Thus, the combination, pharmaceuticalcombination, medicament or kit-of-parts according to the invention is tobe injected to the subject, by intravenous, intramuscular,intraperitoneal, intrapleural, subcutaneous, transdermal injection orinfusion.

In another embodiment, the combination, pharmaceutical combination,medicament or kit-of-parts according to the invention is in an adaptedform for an injection such as, for example, for intravenous,intramuscular, intraperitoneal injection or infusion. Thus, thecombination, pharmaceutical combination, medicament or kit-of-partsaccording to the invention is to be injected to the subject, byintravenous, intramuscular, intraperitoneal, injection or infusion.

Sterile injectable forms of the combination, the pharmaceuticalcombination, medicament or kit-of-parts according to the invention maybe a solution or an aqueous or oleaginous suspension. These suspensionsmay be formulated according to techniques known in the art usingsuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic pharmaceutically acceptable diluent orsolvent. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose, any bland fixed oil may beemployed including synthetic mono- or diglycerides. Fatty acids, such asoleic acid and its glyceride derivatives are useful in the preparationof injectables, as are natural pharmaceutically acceptable oils, such asolive oil or castor oil, especially in their polyoxyethylated versions.These oil solutions or suspensions may also contain a long-chain alcoholdiluent or dispersant, such as carboxymethyl cellulose or similardispersing agents that are commonly used in the formulation ofpharmaceutically acceptable dosage forms including emulsions andsuspensions. Other commonly used surfactants, such as Tweens, Spans andother emulsifying agents or bioavailability enhancers which are commonlyused in the manufacture of pharmaceutically acceptable solid, liquid, orother dosage forms may also be used for the purposes of formulation.

In one embodiment, the CD8 vaccine specific for at least one infectiousdisease-related antigen as described hereinabove is in an adapted formfor a parenteral administration and/or injection. Thus, in anotherembodiment, the CD8 vaccine specific for at least one infectiousdisease-related antigen as described hereinabove is to be administeredparenterally and/or injected to the subject, by intravenous,intramuscular, intraperitoneal, intrapleural, subcutaneous, transdermalinjection or infusion, preferably by intravenous injection.

In another embodiment, the interferon-alpha blocking agent as describedhereinabove is in an adapted form for a parenteral administration and/orinjection. Thus, in another embodiment, the interferon-alpha blockingagent as described hereinabove is to be administered parenterally and/orinjected to the subject, by intravenous, intramuscular, intraperitoneal,intrapleural, subcutaneous, transdermal injection or infusion,preferably by intravenous injection.

In another embodiment, the type III interferon and/or the agentstimulating the production of type III interferon as describedhereinabove is/are in an adapted form for a parenteral administrationand/or injection. Thus, in another embodiment, the type III interferonand/or the agent stimulating the production of type III interferon asdescribed hereinabove is/are to be administered parenterally and/orinjected to the subject, by intravenous, intramuscular, intraperitoneal,intrapleural, subcutaneous, transdermal injection or infusion,preferably by intravenous injection

In another embodiment, the combination, pharmaceutical combination,medicament or kit-of-parts according to the invention is in a formadapted for topical administration. Thus, the combination,pharmaceutical combination, medicament or kit-of-parts according to theinvention is to be administered topically.

Examples of forms adapted for topical administration include, withoutbeing limited to, liquid, paste or solid compositions, and moreparticularly aqueous solutions, drops, dispersions, sprays,microcapsules, micro- or nanoparticles, polymeric patch, orcontrolled-release patch, and the like.

In another embodiment, the CD8 vaccine specific for at least oneinfectious disease-related antigen as described hereinabove is in a formadapted for topical administration. Thus, the CD8 vaccine specific forat least one infectious disease-related antigen as described hereinaboveaccording to the invention is to be administered topically.

In another embodiment, the interferon-alpha blocking agent as describedhereinabove is in a form adapted for topical administration. Thus, theagent interferon-alpha blocking agent as described hereinabove is to beadministered topically.

In another embodiment, the type III interferon and/or the agentstimulating the production of type III interferon as describedhereinabove is/are in a form adapted for topical administration. Thus,the CD8 vaccine specific for at least one infectious disease-relatedantigen as described hereinabove according to the invention is to beadministered topically.

In another embodiment, the combination, pharmaceutical combination,medicament or kit-of-parts according to the invention is in a formadapted for rectal administration. Thus, in one embodiment, thecombination, pharmaceutical combination, medicament or kit-of-partsaccording to the invention is to be to be administered rectally.

Examples of forms adapted for rectal administration include, withoutbeing limited to, suppository, micro enemas, enemas, gel, rectal foam,cream, ointment, and the like.

In another embodiment, the CD8 vaccine specific for at least oneinfectious disease-related antigen as described hereinabove is in a formadapted for rectal administration. Thus, in one embodiment, the CD8vaccine specific for at least one infectious disease-related antigen asdescribed hereinabove is to be to be administered rectally.

In another embodiment, the interferon-alpha blocking agent as describedhereinabove is in a form adapted for rectal administration. Thus, in oneembodiment, the interferon-alpha blocking agent as described hereinaboveis to be to be administered rectally.

In another embodiment, the type III interferon and/or the agentstimulating the production of type III interferon as describedhereinabove is/are in a form adapted for rectal administration. Thus, inone embodiment, the type III interferon and/or the agent stimulating theproduction of type III interferon as described hereinabove is/are to beto be administered rectally.

In one embodiment, the combination, pharmaceutical combination,medicament or kit-of-parts according to the invention comprises 1) a CD8vaccine specific for at least one infectious disease-related antigen, 2)optionally an interferon-alpha blocking agent, and 3) a type IIIinterferon and/or an agent stimulating the production of type IIIinterferon which are all in a form adapted for parenteral administrationand/or injection.

In one embodiment, the combination, pharmaceutical combination,medicament or kit-of-parts according to the invention comprises 1) a CD8vaccine specific for at least one infectious disease-related antigen, 2)optionally an interferon-alpha blocking agent, and 3) a type IIIinterferon and/or an agent stimulating the production of type IIIinterferon which are all in a form adapted for oral administration.

In another embodiment, the combination, pharmaceutical combination,medicament or kit-of-parts according to the invention comprises 1) a CD8vaccine specific for at least one infectious disease-related antigenthat is in a form adapted for oral administration, and 2) optionally aninterferon-alpha blocking agent, and 3) a type III interferon and/or anagent stimulating the production of type III interferon that is/are in aform adapted for parenteral administration and/or injection.

In another embodiment, the combination, pharmaceutical combination,medicament or kit-of-parts according to the invention comprises 1) a CD8vaccine specific for at least one infectious disease-related antigenthat is in a form adapted for oral administration, and 2) optionally aninterferon-alpha blocking agent, and 3) a type III interferon and/or anagent stimulating the production of type III interferon that is/are in aform adapted for parenteral administration and/or injection.

In another embodiment, the combination, pharmaceutical combination,medicament or kit-of-parts according to the invention comprises 1) a CD8vaccine specific for at least one infectious disease-related antigenthat is in a form adapted for rectal administration, and 2) optionallyan interferon-alpha blocking agent, and 3) a type III interferon and/oran agent stimulating the production of type III interferon that is/arein a form adapted for parenteral administration and/or injection.

In another embodiment, the combination, pharmaceutical combination,medicament or kit-of-parts according to the invention comprises 1) a CD8vaccine specific for at least one infectious disease-related antigenthat is in a form adapted for vaginal administration, and 2) optionallyan interferon-alpha blocking agent, and 3) a type III interferon and/oran agent stimulating the production of type III interferon that is/arein a form adapted for parenteral administration and/or injection.

As mentioned hereinabove, the administration of each part of thecombination, pharmaceutical combination, medicament or kit-of-partsaccording to the invention can be done simultaneously, separately orsequentially.

In one embodiment, the combination, pharmaceutical combination,medicament or kit-of-parts according to the invention comprises a firstpart comprising a CD8 vaccine specific for at least one infectiousdisease-related antigen, optionally a second part comprising aninterferon-alpha blocking agent, and a third part comprising a type IIIinterferon and/or an agent stimulating the production of type IIIinterferon which are all administered at the same time once, twice,three, four, five, six, seven, eight, nine, ten times or more.

In one embodiment of the invention, the first part of the combination,pharmaceutical combination or kits of parts is to be administered priorto the second part and the third part of the combination, pharmaceuticalcombination or kits of parts once, twice, three, four, five, six, seven,eight, nine, ten times or more.

In another embodiment of the invention, the second part of thecombination, pharmaceutical combination or kits of parts is to beadministered prior to the first part and the third part of thecombination, pharmaceutical combination or kits of parts once, twice,three, four, five, six, seven, eight, nine, ten times or more.

In another embodiment of the invention, the third part of thecombination, pharmaceutical combination or kits of parts is to beadministered prior to the first part and the second part of thecombination, pharmaceutical combination or kits of parts once, twice,three, four, five, six, seven, eight, nine, ten times or more.

In one embodiment of the invention, the first part and the second partof the combination, pharmaceutical combination or kits of parts are tobe administered prior to the third part of the combination,pharmaceutical combination or kits of parts once, twice, three, four,five, six, seven, eight, nine, ten times or more.

In another embodiment of the invention, the first part and the thirdpart of the combination, pharmaceutical combination or kits of parts isto be administered prior to the second part of the combination,pharmaceutical combination or kits of parts once, twice, three, four,five, six, seven, eight, nine, ten times or more.

In another embodiment of the invention, the second part and the thirdpart of the combination, pharmaceutical combination or kits of parts isto be administered prior to the first part and the second part of thecombination, pharmaceutical combination or kits of parts once, twice,three, four, five, six, seven, eight, nine, ten times or more.

In one embodiment of the invention, the first part and the second partof the combination, pharmaceutical combination or kits of parts are tobe administered at the same time once, twice, three, four, five, six,seven, eight, nine, ten times or more.

In one embodiment of the invention, the first part and the third part ofthe combination, pharmaceutical combination or kits of parts are to beadministered at the same time once, twice, three, four, five, six,seven, eight, nine, ten times or more.

In one embodiment of the invention, the second part and the third partof the combination, pharmaceutical combination or kits of parts are tobe administered at the same time once, twice, three, four, five, six,seven, eight, nine, ten times or more.

In one embodiment of the invention, the first part of the combination,pharmaceutical combination or kits of parts is to be administered priorto the second part of the combination, pharmaceutical combination orkits of parts once, twice, three, four, five, six, seven, eight, nine,ten times or more, and the second part is to be administered prior tothe third part of the combination, pharmaceutical combination or kits ofparts once, twice, three times or more.

In another embodiment of the invention, the second part of thecombination, pharmaceutical combination or kits of parts is to beadministered prior to the first part of the combination, pharmaceuticalcombination or kits of parts once, twice, three, four, five, six, seven,eight, nine, ten times or more, and the first part is to be administeredprior to the third part of the combination, pharmaceutical combinationor kits of parts once, twice, three, four, five, six, seven, eight,nine, ten times or more.

In one embodiment of the invention, the second part of the combination,pharmaceutical combination or kits of parts is to be administered priorto the third part of the combination, pharmaceutical combination or kitsof parts once, twice, three, four, five, six, seven, eight, nine, tentimes or more, and the third part is to be administered prior to thefirst part of the combination, pharmaceutical combination or kits ofparts once, twice, three, four, five, six, seven, eight, nine, ten timesor more.

In one embodiment of the invention, the third part of the combination,pharmaceutical combination or kits of parts is to be administered priorto the first part of the combination, pharmaceutical combination or kitsof parts once, twice, three times, four, five, six, seven, eight, nine,ten or more, and the first part is to be administered prior to thesecond part of the combination, pharmaceutical combination or kits ofparts once, twice, three, four, five, six, seven, eight, nine, ten timesor more.

In one embodiment of the invention, the third part of the combination,pharmaceutical combination or kits of parts is to be administered priorto the second part of the combination, pharmaceutical combination orkits of parts once, twice, three, four, five, six, seven, eight, nine,ten times or more, and the second part is to be administered prior tothe first part of the combination, pharmaceutical combination or kits ofparts once, twice, three, four, five, six, seven, eight, nine, ten timesor more.

In another embodiment, the combination, pharmaceutical combination,medicament or kit-of-parts according to the invention comprises a firstpart comprising a CD8 vaccine specific for at least one infectiousdisease-related antigen, optionally a second part comprising aninterferon-alpha blocking agent, and a third part comprising a type IIIinterferon and/or an agent stimulating the production of type IIIinterferon which are all administered at the same time once a day for 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more days.

In one embodiment, the combination, pharmaceutical combination,medicament or kit-of-parts according to the invention comprises a firstpart comprising a CD8 vaccine specific for at least one infectiousdisease-related antigen, optionally a second part comprising aninterferon-alpha blocking agent, and a third part comprising a type IIIinterferon and/or an agent stimulating the production of type IIIinterferon which are all administered at the same time once a month for1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more months.

In one embodiment, the combination, pharmaceutical combination,medicament or kit-of-parts according to the invention comprises a firstpart comprising a CD8 vaccine specific for at least one infectiousdisease-related antigen, optionally a second part comprising aninterferon-alpha blocking agent, and/or a third part comprising a typeIII interferon and an agent stimulating the production of type IIIinterferon which are all administered at the same time once a year for1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years.

In one embodiment, the administration of each part of the combination,pharmaceutical combination, medicament or kit-of-parts according to theinvention can be done at the same time or at different time.

Another object of the invention is a method for preventing or treatingan infectious disease in a subject comprising administering to thesubject in need thereof a therapeutically effective amount of:

-   -   1) a CD8 vaccine specific for at least one infectious        disease-related antigen,    -   2) optionally an interferon-alpha blocking agent, and    -   3) a type III interferon and/or an agent stimulating the        production of type III interferon.

According to one embodiment, a therapeutically effective dose of thefirst part of the combination, pharmaceutical combination, medicament orkit-of-parts a as described hereinabove is to be administered incombination with a therapeutically effective dose of the second part ofthe combination, pharmaceutical combination, medicament or kit-of-partsas described hereinabove and a therapeutically effective dose of thethird part of the combination, pharmaceutical combination, medicament orkit-of-parts as described hereinabove for use in the treatment of aninfectious disease in a subject in need thereof. Thus, in oneembodiment, the combination, pharmaceutical combination, medicament orkit-of-parts according to the invention comprises a therapeuticallyeffective dose of first part as described hereinabove and atherapeutically effective dose of second part as described hereinaboveand a therapeutically effective dose of third part as describedhereinabove described hereinabove.

According to one embodiment, a therapeutically effective dose of thefirst part of the combination, pharmaceutical combination, medicament orkit-of-parts a as described hereinabove is to be administered incombination with a therapeutically effective dose of the second part ofthe combination, pharmaceutical combination, medicament or kit-of-partsas described hereinabove for use in the treatment of an infectiousdisease in a subject in need thereof. Thus, in one embodiment, thecombination, pharmaceutical combination, medicament or kit-of-partsaccording to the invention comprises a therapeutically effective dose offirst part as described hereinabove and a therapeutically effective doseof second part as described hereinabove.

According to one embodiment, a therapeutically effective dose of thefirst part of the combination, pharmaceutical combination, medicament orkit-of-parts a as described hereinabove is to be administered incombination with a therapeutically effective dose of the third part ofthe combination, pharmaceutical combination, medicament or kit-of-partsas described hereinabove for use in the treatment of an infectiousdisease in a subject in need thereof. Thus, in one embodiment, thecombination, pharmaceutical combination, medicament or kit-of-partsaccording to the invention comprises a therapeutically effective dose ofthe first part as described hereinabove and a therapeutically effectivedose of third part as described hereinabove.

In one embodiment, the administration of each part of the combination,pharmaceutical combination, medicament or kit-of-parts according to theinvention can be done according to a prime/boost mode. Thus, the presentinvention also includes a variety of prime-boost regimens.

In one embodiment, the prime/boost mode comprises the steps ofadministrating:

-   -   one or more priming immunizations, wherein said boosting        immunizations comprises: a therapeutically effective dose of the        first part, a therapeutically effective dose of the second part,        and/or a therapeutically effective dose of the third part, and    -   one or more boosting immunizations.

In prime/boost regimens, the composition of each part of thecombination, according to the invention may be the same or different foreach immunization and the type of composition, the route, andformulation of each part of the combination pharmaceutical combination,medicament or kit-of-parts according to the invention may also bevaried. For example, if an expression vector is used for the priming andboosting steps, it may either be of the same or different type (e.g.,DNA or bacterial or viral expression vector). For example, one usefulprime-boost regimen provides for at least two priming immunizations, twoweeks apart, followed by at least one boosting immunizations (e.g., at4-5 and/or 8-9 weeks) after the last priming immunization. It shouldalso be readily apparent to one of skill in the art that there areseveral permutations and combinations that are encompassed using theDNA, bacterial and viral expression vectors or bacteria of thedisclosure to provide priming and boosting regimens. For example, CMVvectors may be used repeatedly while expressing different antigensderived from the same or different pathogens.

In one embodiment, the boosting immunization step comprises theadministration of a non-infectious dose of SIV or HIV, or an attenuatedSIV or HIV (e.g., HIV or SIV depleted in protein net). In oneembodiment, the boosting immunization is in a form adapted for oral,rectal or vaginal administration.

Attenuated SIV or HIV virus are well known in the art. A non limitatingexample of said attenuated virus is an HIV or SIV depleted in proteinnef (see, for example, Giorgi et al., J Med Primatol. 1996 June;25(3):186-91).

In some embodiments, the second part and/or the third part of thecombination, pharmaceutical combination, medicament or kit-of-partsaccording to the invention are to be administered at time and route ofadministration separately from the first part.

In one embodiment, the first part of the combination, pharmaceuticalcombination, medicament or kit-of-parts according to the invention is tobe administered at least 2 times (e.g., at days 0 and 14). In anotherembodiment, the second part and/or the third part of the combination,pharmaceutical combination, medicament or kit-of-parts according to theinvention are to be administered at least 2 times before theadministration of the first (e.g., at days −7 and day −3), and at least9 times after the administration (e.g., at days 3, 11, 38, 45, 52, 59,66, 73 and 80).

In one embodiment, the first part of the combination, pharmaceuticalcombination, medicament or kit-of-parts according to the invention is tobe administered at least 7 times (e.g., at days 0, 1, 3, 7, 28 and 29).In another embodiment, the second part and/or the third part of thecombination, pharmaceutical combination, medicament or kit-of-partsaccording to the invention are to be administered before administrationsof the first part (e.g., at days −3, 0, 28 and 29), and at least 1 moretime (e.g., at days 57).

In one embodiment, the first part of the combination, pharmaceuticalcombination, medicament or kit-of-parts according to the invention is tobe administered at least 7 times during the priming and boost steps(e.g., at days 0, 1, 2, 3, 5 for priming and days 28 and 29 for thefirst boost). The second part of the combination, pharmaceuticalcombination, medicament or kit-of-parts according to the invention is tobe administered at least one time during the priming and first booststeps (e.g. from day 0 to 40) and at least one time after the lastadministration of the first part of the combination, pharmaceuticalcombination, medicament or kit-of-parts according to the invention. Thethird part of the combination, pharmaceutical combination, medicament orkit-of-parts according to the invention is to be administered 0, 1, 2 or3 days before each administration of the first part of the combination,pharmaceutical combination, medicament or kit-of-parts according to theinvention.

In one embodiment, the prime/boost mode comprises a step ofadministrating a non-infectious dose of SIV or HIV, or of an attenuatedSIV or HIV (e.g., at day 60).

It will be understood that the total daily usage of the first part, thetotal daily usage of the second part and the total daily usage of thirdpart in the combination, the pharmaceutical combination, medicament orkit-of-parts according to the invention will be decided by the attendingphysician within the scope of sound medical judgment. The specific dosefor any particular subject will depend upon a variety of factors such asthe infectious disease to be treated; the age, body weight, generalhealth, sex and diet of the subject, and like factors well-known in themedical arts. Hence, the combination, the pharmaceutical combination,medicament or kit-of-parts according to the invention can beadministered one or more times to the subject. Preferably, there is aset time interval between separate administrations of the combination,the pharmaceutical combination, medicament or kit-of-parts according tothe invention. While this interval varies for every subject, typicallyit ranges from 1 days to several weeks, and is often 1, 2, 4, 6 or 8days, or 1, 2, 4, 6 or 8 weeks. In one embodiment of the presentinvention, the interval is typically from 1 to 6 weeks. In oneembodiment of the present invention, the interval is longer,advantageously about 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks,20 weeks, 22 weeks, 24 weeks, 26 weeks, 28 weeks, 30 weeks, 32 weeks, 34weeks, 36 weeks, 38 weeks, 40 weeks, 42 weeks, 44 weeks, 46 weeks, 48weeks, 50 weeks, 52 weeks, 54 weeks, 56 weeks, 58 weeks, 60 weeks, 62weeks, 64 weeks, 66 weeks, 68 weeks 70 weeks or 80 weeks. In oneembodiment, the administration regimes typically have from 1 to 20administrations of the 3 different parts according to the invention, butmay have as few as one or two or four or eight or ten. In anotherembodiment the administration regimes is annual, biannual or other longinterval (5-10 years).

In one embodiment, the subject is a mammal, a primate, preferably ahuman.

As an example, when the first part of the pharmaceutical combination,medicament or kit-of-parts according to the invention comprises a CMVvector as described herein above, and when the subject to be treated isa mammal, a primate or a human, the therapeutically effective dose ofsaid CMV vector can range from a few to a few hundred micrograms (e.g.,5 to 500 μg per administration). The CMV vector can be administrated inany suitable amount to achieve expression at these dosage levels. Innon-limiting examples, CMV vectors may be administered in an amount ofat least 10¹, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷ or 10⁸ pfu peradministration. Thus, CMV vectors may be administered in at least 10¹pfu, or in a range from about 10¹ pfu to about 10⁸ pfu peradministration. The CMV vector may be lyophilized for resuspension atthe time of administration or may be in solution.

In one embodiment, the amount of CMV vectors, as described hereinabove,administered to the subject is at least of 10¹, 10², 10³, 10⁴, 10⁵, 10⁶,10⁷ or 10⁸ pfu. In one embodiment, the amount of CMV vectors, asdescribed hereinabove, administered per administration ranges from about10¹ to about 10⁸, preferably from about 10² to about 10⁷, morepreferably from about 10³ to about 10⁶, and even more preferably fromabout 10⁴ to about 10⁵, including all integer values within thoseranges. In one embodiment, the daily amount of CMV vectors, as describedhereinabove, administered per day to the subject is at least of 10¹ perday, 10² per day, 10³ per day, 10⁴ per day, 10⁵ per day, 10⁶ per day,10⁷ per day, 10⁸ per day of pfu. In one embodiment, the daily amount ofCMV vectors, as described hereinabove, administered per day ranges fromabout 10¹ to about 10⁸ per day, preferably from about 10² to about 10⁷per day, more preferably from about 10³ to about 10⁶ per day, and evenmore preferably from about 10⁴ to about 10⁵ per day, including allinteger values within those ranges. In one embodiment, the amount of CMVvectors, as described hereinabove, administered to the subject is atleast of 10¹, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷ or 10⁸ viruses/kg body.

As an example, when the first part of the pharmaceutical combination,medicament or kit-of-parts according to the invention comprises at leastone infectious disease-related antigen and a non-pathogenic bacterium asdescribed herein above, and when the subject to be treated is a human,the therapeutically effective dose of said non-pathogenic bacteria(i.e., Lactobacillus sp. or Lactobacillus plantarum) can range fromabout 10¹ to about 10¹⁸ cfu per administration and the therapeuticallyeffective dose of said infectious disease-related antigen (i.e.,inactivated SIV or HIV viruses) can range from about 10¹ to about 10¹⁴viruses per administration.

In one embodiment, the amount of non-pathogenic bacteria, as describedhereinabove, administered to the subject is at least of 10¹, 10², 10³,10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³ or 10¹⁴ cfu. In oneembodiment, the amount of non-pathogenic bacteria, as describedhereinabove, administered per administration ranges from about 10¹ toabout 10¹⁸, preferably from about 10² to about 10¹⁶, more preferablyfrom about 10⁴ to about 10¹⁴, and even more preferably from about 10⁶ toabout 10¹², including all integer values within those ranges. In oneembodiment, the daily amount of non-pathogenic bacteria, as describedhereinabove, administered per day to the subject is at least of 10¹ perday, 10² per day, 10³ per day, 10⁴ per day, 10⁵ per day, 10⁶ per day,10⁷ per day, 10⁸ per day, 10⁹ per day, 10¹⁰ per day, 10¹¹ per day, 10¹²per day, 10¹³ per day, 10¹⁴ per day, 10¹⁵ per day, 10¹⁶ per day, 10¹⁷per day or 10¹⁸ per day of cfu. In one embodiment, the daily amount ofnon-pathogenic bacteria, as described hereinabove, administered per dayranges from about 10¹ to about 10¹⁸ per day, preferably from about 10²to about 10¹⁶ per day, more preferably from about 10⁴ to about 10¹⁴ perday, and even more preferably from about 10⁶ to about 10¹² per day,including all integer values within those ranges. In one embodiment, theamount of non-pathogenic bacteria, as described hereinabove,administered to the subject is at least of 10¹, 10², 10³, 10⁴, 10⁵, 10⁶,10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³ or 10¹⁴ bacteria/kg body.

In one embodiment, the amount of inactivated SIV or HIV viruses, asdescribed hereinabove, administered to the subject is at least of 10¹,10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, or 10¹⁴viruses. In one embodiment, the amount of inactivated SIV or HIVviruses, as described hereinabove, administered per administrationranges from about 10¹ to about 10¹⁸, preferably from about 10² to about10¹⁶, more preferably from about 10⁴ to about 10¹⁴, and even morepreferably from about 10⁶ to about 10¹², including all integer valueswithin those ranges. In one embodiment, the daily amount of inactivatedSIV or HIV viruses, as described hereinabove, administered per day tothe subject is at least of 10¹ per day, 10² per day, 10³ per day, 10⁴per day, 10⁵ per day, 10⁶ per day, 10⁷ per day, 10⁸ per day, 10⁹ perday, 10¹⁰ per day, 10¹¹ per day, 10¹² per day, 10¹³ per day, 10¹⁴ perday, 10¹⁵ per day, 10¹⁶ per day, 10¹⁷ per day or 10¹⁸ per day ofviruses. In one embodiment, the daily amount of inactivated SIV or HIVviruses, as described hereinabove, administered per day ranges fromabout 10¹ to about 10¹⁸ per day, preferably from about 10² to about 10¹⁶per day, more preferably from about 10⁴ to about 10¹⁴ per day, and evenmore preferably from about 10⁶ to about 10¹² per day, including allinteger values within those ranges. In one embodiment, the amount ofinactivated SIV or HIV viruses, as described hereinabove, administeredto the subject is at least of 10¹, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸,10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, or 10¹⁴⁼ viruses/kg body.

In one embodiment, the subject is a mammal, a primate, preferably ahuman, and said therapeutically effective dose of the first part of thecombination, pharmaceutical combination medicament or kit-of-partsaccording to the invention is a daily dose to be administered in one,two, three or more takes or in one, two, three or more injections

In one embodiment, the subject is a mammal, a primate, preferably ahuman, and said therapeutically effective dose of the second part of thecombination, pharmaceutical combination medicament or kit-of-partsaccording to the invention is a daily dose to be administered in one,two, three or more takes or in one, two, three or more injections.

In one embodiment, the subject is a mammal, a primate, preferably ahuman, and said therapeutically effective dose of the third part of thecombination, pharmaceutical combination medicament or kit-of-partsaccording to the invention is a daily dose to be administered in one,two, three or more takes or in one, two, three or more injections.

According to the present invention, the combination, pharmaceuticalcombination, medicament or kit-of-parts as described hereinabove is usedalone.

Thus, in one embodiment, the combination, pharmaceutical combination,medicament or kit-of-parts according to the invention is used alone andcomprises a therapeutically effective dose of first part as describedhereinabove and a therapeutically effective dose of second part asdescribed hereinabove and a therapeutically effective dose of third partas described hereinabove described hereinabove. In one embodiment, thecombination, pharmaceutical combination, medicament or kit-of-partsaccording to the invention is used alone and comprises a therapeuticallyeffective dose of first part as described hereinabove and atherapeutically effective dose of second part as described hereinabove.In another embodiment, the combination, pharmaceutical combination,medicament or kit-of-parts according to the invention is used alone andcomprises a therapeutically effective dose of first part as describedhereinabove and a therapeutically effective dose of third part asdescribed hereinabove

In one embodiment, the present invention, the combination,pharmaceutical combination, medicament or kit-of-parts as describedhereinabove is used in combination with at least one further therapeuticagent.

Such administration may be simultaneous, separate or sequential. Forsimultaneous administration the agents may be administered as onecomposition or as separate compositions, as appropriate. The furthertherapeutic agent is typically relevant for disorders to be treated.

Thus, in one embodiment, the combination, pharmaceutical combination,medicament or kit-of-parts according to the invention comprises atherapeutically effective dose of first part as described hereinaboveand a therapeutically effective dose of second part as describedhereinabove and a therapeutically effective dose of third part asdescribed hereinabove described hereinabove, and is used in combinationwith at least one further therapeutic agent. In one embodiment, thecombination, pharmaceutical combination, medicament or kit-of-partsaccording to the invention comprises a therapeutically effective dose offirst part as described hereinabove and a therapeutically effective doseof second part as described hereinabove, and is used in combination withat least one further therapeutic agent. In another embodiment, thecombination, pharmaceutical combination, medicament or kit-of-partsaccording to the invention comprises a therapeutically effective dose offirst part as described hereinabove and a therapeutically effective doseof third part as described hereinabove is used in combination with atleast one further therapeutic agent.

In one embodiment, the further therapeutic agent is an antiretroviraltherapy (ART).

As used herein, the term “antiretroviral therapy” or “highly activeantiretroviral therapy” refers to any combination of antiretroviral(ARV) drugs to maximally suppress the HIV virus (e.g., reduce viral loadreduce HIV multiplication . . . ), and stop the progression of HIVdisease. There are several classes of HIV drug, such as, for example,non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleosidereverse transcriptase inhibitors (NRTIs), post-attachment inhibitors,protease inhibitors (PIs), CCR5 antagonists, integrase strand transferinhibitors (INSTIs), fusion inhibitors. Generally, initial treatmentregimens usually include two NTRIs combined with a third activeantiretroviral drug, which may be in the INSTI, NNRTI, or PI class. Theymay sometimes include a booster, which may be cobicistat (Tybost) orritonavir (Norvir).

In one embodiment, the combination, pharmaceutical combination,medicament or kit-of-parts as described hereinabove is used incombination with an antiretroviral therapy (ART).

Thus, in one embodiment, the combination, pharmaceutical combination,medicament or kit-of-parts according to the invention comprises atherapeutically effective dose of first part as described hereinaboveand a therapeutically effective dose of second part as describedhereinabove and a therapeutically effective dose of third part asdescribed hereinabove described hereinabove, and is used in combinationwith an antiretroviral therapy. In one embodiment, the combination,pharmaceutical combination, medicament or kit-of-parts according to theinvention comprises a therapeutically effective dose of first part asdescribed hereinabove and a therapeutically effective dose of secondpart as described hereinabove, and is used in combination with anantiretroviral therapy. In another embodiment, the combination,pharmaceutical combination, medicament or kit-of-parts according to theinvention comprises a therapeutically effective dose of first part asdescribed hereinabove and a therapeutically effective dose of third partas described hereinabove is used in combination with an antiretroviraltherapy.

According to the present invention, the combination pharmaceuticalcombination medicament or kit-of-parts as described hereinabove is foruse in the prevention or treatment of an infectious disease in a subjectin need thereof.

As used herein, the term “infectious disease” refers to a disease causedby a pathogen, such as a fungus, parasite, protozoan, bacterium orvirus. Examples of “infectious diseases” include, without being limitedto, influenza virus infection, monkeypox virus infection, West Nilevirus infection, Chikungunya virus infection, Ebola virus infection,hepatitis C virus infection, poliovirus infection, dengue fever,Acquired immune deficiency syndrome (AIDS) or a Simian Immunodeficiencyvirus (SIV) infection and the recombinant RhCMV or HCMV vector encodesan antigen from HIV or SIV, skin warts, genital warts, respiratorypapillomatosis, Malaria, Ebola hemorrhagic fever, Tuberculosis, Herpesdisease (e.g., Genital Herpes, Chicken pox or Herpes Zoster, Infectiousmononucleosis), tuberculosis infection (caused by Mycobacteriumtuberculosis), typhoid infection or fever (caused by Salmonella typhi).

In one embodiment, the infectious disease to be prevented or treated ispreferably acquired immune deficiency syndrome (AIDS), a humanimmunodeficiency virus (HIV) infection or a simian immunodeficiencyvirus (SIV) infection.

In one embodiment, the infectious disease to be prevented or treated isacquired immune deficiency syndrome (AIDS).

In some embodiments, the combination pharmaceutical combinationmedicament or kit-of-parts as described hereinabove is for use in theprophylactic treatment or in the curative treatment of an infectiousdisease in a subject in need thereof.

EXAMPLES

The present invention is further illustrated by the following examples.

Example 1: Effects of Type I and Type III Interferons on Innate andAdaptative Immune Responses Materials and Methods Human Cell Lines

HCC HepG2 and normal kidney epithelial Vero cell lines were obtainedfrom ATCC. Cells were grown in Dulbecco's Modified Eagle Mediumsupplemented with 10% heat-inactivated Fetal Bovine Serum, 2 mML-glutamine, 1% penicillin and streptomycin solution in hypoxia 2%.Cancer cell lines were grown to 70-100% confluency, subsequentlypassaged for a maximum of 5 times and freshly thawed thereafter. Cellswere detached by means of accutase, resuspended in FBS-containing mediumand collected by means of centrifugation (300 g, 3 min). Cell numberswere determined by means of trypan blue.

Human Blood Sample

Blood samples from healthy individuals originated from EtablissementFrancais du Sang (EFS, Paris). Blood cells are collected using standardprocedures.

Cell Purification and Culture

Peripheral blood mononuclear cells (PBMCs) are isolated by densitygradient centrifugation on Ficoll-Hypaque (Pharmacia). PBMCs are usedeither as fresh cells or stored frozen in liquid nitrogen. T-cellsubsets and T cell-depleted accessory cells (ΔCD3 cells) are isolatedfrom either fresh or frozen PBMCs. T cell-depleted accessory cells (ΔCD3cells) are isolated by negative selection from PBMCs by incubation withanti-CD3-coated Dynabeads (Dynal Biotech) and are irradiated at 3000 rad(referred to as ΔCD3-feeder). CD4⁺ T cells are negatively selected fromPBMCs with a CD4⁺ T-cell isolation kit (Miltenyi Biotec), yielding CD4⁺T-cell populations at a purity of 96-99%. T cell subsets are culturedeither in IMDM supplemented with 5% SVF, 100 IU/mlpenicillin/streptomycin, 1 mM sodium pyruvate, 1 mM nonessential aminoacids, glutamax and 10 mM HEPES (IMDM-5 media) in hypoxia 2%.

Freezing and Thawing of Cells

Cells were frozen in FBS containing 10% DMSO. Cryo tubes were placed inCoolCell (Biocision) freezing containers and incubated at −80° C. After2 days tubes were transferred to liquid nitrogen and stored untilrequired. Thawing of cells was performed by placing cryo tubes in a 37°C. water bath for approximately 30 seconds. Next, cell suspension wasmixed with equivalent volume of pre-warmed media and subsequentlytransferred to falcon tubes containing the same medium. Cells werepelleted by centrifugation (300 g, 3 min) to remove DMSO. The cellpellet was resuspended in cell culture medium

Real-Time PCR for ISGs Detection

HepG2 cells were seeded at a density of 2×10⁵ cells per well in 12-wellplates and incubated for 24 h. Then, fresh media was added with theindicated interferons. The cells were incubated for 4 h and then lysed,and RNA was purified using an extraction kit (Qiagen), according to themanufacturer's instructions. Synthesis of cDNA was performed using thePrimeScript RT Reagent kit (TAKARA). Quantitative PCR was carried outusing the Power SYBR Green PCR Master Mix (Applied Biosystems) on aLightCycler 480 instrument (Roche). Each reaction was carried out induplicate in a total volume of 100 μL. Primers were designed to beintron-spanning using Primer3 or Primer Express® v3.0 software (AppliedBiosystems). To measure the cellular transcriptional response to IFNstimulation, 3 ISG targets, MXI, OASL and ISG15, were selected based onpublished results investigating the transcriptional response inIFN-stimulated PBMCs (see, for example, Waddell et al. (2010) PLoS One.5(3):e97532). For gene induction assays, fold change values werecalculated using the AACt method. The geometric mean of the Ct values ofthe reference genes, S14, was used as a reference value.

Virus Production

The virus used EMCV (FA strain) was grown on monolayers of Vero cells tocomplete cytopathic effect or until all cells were affected by theinfection as determined by microscopy and prepared by two cycles offreezing and thawing, followed by centrifugation for 30 min at 5,000×gfor removal of cellular debris.

Antiviral Assay

Antiviral assays were done on HepG2 cells, which were seeded in DMEMsupplemented with 10% FCS at a density of 1.5×10⁴ in 96-well plates andleft to settle. The cells were incubated with indicated doses of IFNsfor 24 h before challenge with EMCV. The cells were incubated with virusfor 48 h. The medium was removed between each step. The viability of thecells was analyzed by a bioassay based on the dehydrogenase system; thissystem in intact cells will convert the substrate, MTT, into formazan(blue), which in turn can be measured spectrophotometrically. Briefly,the cells were given MTT and incubated for 2 h. An extraction buffer(containing 6 to 11% sodium dodecyl sulfate and 45%N,N-dimethylformamide) was added to the cells, and the cells were thenincubated overnight at 37° C. Subsequently, the absorbance at 570 nm wasdetermined employing the extraction buffer as the blank probe. A570 wasdirectly proportional to antiviral activity.

Flow Cytometry Analysis

CD3⁺ T cells staining: anti-CD4 (SK3)-APC, anti-CD3 (UCHT1)-FITC,anti-CD8 (RPA-T8)-BV421 are from Becton Dickinson. Cells are stained forsurface markers (at 4° C. in the dark for 30 min) using mixtures of Abdiluted in PBS containing 3% FBS, 2 mM EDTA (FACS buffer).

STAT1 signaling analysis: Flow cytometry analysis of STAT1phosphorylation (pSTAT1) was conducted in CD4⁺ T cells by using BDPhosflow technology according to the manufacturer's instructions (BDBio-sciences, San Jose, Calif.). CD4⁺ T cells were stimulated byincubation with interferon type I and Type III at 37° C. for 20 min orleft untreated. Activation was stopped by fixation using BD PhosflowLyse/Fix Buffer (BD Biosciences) and cells were permeabilized with BDPerm Buffer III (BD Biosciences). Cells were stained with antibodyrecognizing specific phosphorylated STAT tyrosines: p-STAT1 (Y701)-PE.In multiparametric immunophenotyping experiments, cells weresimultaneously stained with anti-CD3-FITC and 7-AAD. Increases in pSTAT1were assayed as a ratio of induction over baseline levels (MFI foldchange=MFI cytokine-stimulated/MFI untreated cells)

CFSE staining: CD4⁺ T cells were stained with 1 μM CFSE (CellTrace cellproliferation kit; Molecular Probes/Invitrogen) in PBS for 8 min at 37°C. at a concentration of 1.107 cells/ml. The labeling reaction wasstopped by washing twice the cell with RPMI-1640 culture mediumcontaining 10% 1-.BS. The cells were then re-suspended at the desiredconcentration and subsequently used for proliferation assays.

7-AAD staining: Apoptosis of stimulated CFSE-labeled CD4⁺ T wasdetermined using the 7-AAD assay. Briefly, cultured cells were stainedwith 20 μg/mL nuclear dye 7-amino-actinomycin D (7-AAD; Sigma-Aldrich,St-Quentin Fallavier, France) for 30 minutes at 4° C. FSC/7-AAD dotplots distinguish living (FSC^(high)/7-AAD⁻) from apoptotic(FSC^(high)/7-AAD⁺) cells and apoptotic bodies (FSC^(low)/7-AAD⁺) anddebris ((FSC^(low)/7-AAD⁻). Living cells were identified as CD3⁺7-AAD-FSC⁺ cells.

Appropriate isotype control Abs are used for each staining combination.Samples are acquired on a BD LSR FORTESSA flow cytometer using BDFACSDIVA 8.0.1 software (Becton Dickinson). Results are expressed inpercentage (%) or in mean fluorescence intensity (MFI).

Functionnal Assay

T cell proliferation: T cell proliferation was assessed withCFSE-dilution assays. For CFSE-dilution assay, at coculture completion,stimulated CFSE-labeled CD4⁺ T cells were harvested, co-stained withanti-CD3 mAb and 7-AAD, and the percentage of proliferating cells(defined as CFSE low fraction) in gated CD3⁺ 7-AAD⁻ cells was determinedby flow cytometry.

T cell activation: CD38 Median Fluorescence Intensity (MFI) of CD38expression was measured by flow cytometry in CD3⁺ 7-AAD-CFSE⁺ stimulatedCD4⁺ T cells at the end of the culture.

CD4⁺ T cell polyclonal stimulation: CFSE-stained CD4⁺ T cells(5×10⁴/well) were cultured in 96 round-bottomed microwells in thepresence of ΔCD3-feeder (1×10⁵/well) and plate-bound anti-CD3 Ab (2μg/ml), soluble anti-CD28 mAb (2 μg/ml). CD4⁺ T cell proliferation wasevaluated with CFSE dilution assays as described above by flowcytometry. Cells were stimulated in presence of different amounts ofrecombinant cytokines.

Allogeneic mixed lymphocyte reaction: CFSE-stained CD4⁺ T cells(5×10⁴/well) were cultured in 96 round-bottomed microwells in thepresence of allogeneic mature DC. Proliferation of allo-activated CD4⁺ Tcells with CFSE dilution assays as described above by flow cytometry.Cells were stimulated in presence of different amounts of recombinantcytokines.

Stat1 phosphorylation analysis: CD4⁺ T cells were stimulated withIFN-λ1, IFN-λ2, IFN-λ3, IFN-λ4, or IFN-a2a (10 ng/ml) for 20 min, orwere left unstimulated (control). Phosphorylated Stat1 levels wasassessed by flow cytometry as described above.

Results

Type I interferons (IFN-α/β) and the more recently identified type IIIIFNs (IFN-λ) function as the first line of defense against virusinfection, and regulate the development of both innate and adaptiveimmune responses. Type III IFNs were originally identified as a novelligand-receptor system acting in parallel with type I IFNs, butsubsequent studies have provided increasing evidence for distinct rolesfor each IFN family.

The inventors aimed to evaluate the effects of type I and type IIIinterferons on both innate (antiviral) and adaptive immune response(CD4⁺ T cell proliferation).

Antiviral Activities of Types I and III

The ability of IFN type I and III to induce the expression ofinterferon-stimulated genes (ISGs) was analyzed by qPCR.

Briefly, the antiviral activity of type I and III was tested in HepG2cells treated with IFN-α2a, IFNλ1, IFNλ2, IFNλ3 or IFNλ4 for 4 hours.Then the induction of the well-known interferon-stimulated genes (ISGs)MX1, IFIT1 and OASL was monitored by qPCR.

As shown in FIG. 1A, all five interferons clearly induced all threeISGs.

Since the investigated ISGs are functionally related to an antiviraldefense, the inventors further evaluate the capacity of both IFN toprotect HepG2 cells from EMCV-induced cytopathogenic effect.

Briefly, cells were seeded in a 96-well microtiter plate and treatedwith the indicated amount of IFNs for 24 h and then challenged with EMCVfor 20 h. Cell survival was measured by an MTT coloring assay.

As shown in FIG. 1B, IFN type III and IFN-α2a have intrinsic cellularantiviral activity and are able to fully protect HepG2 cells challengedwith EMCV.

Anti Proliferative Activity of Type I and Type III Interferons AgainstCD4⁺ T Cells Proliferation

The effect of IFN-type I and IFN type III on CD4⁺ T cells proliferationin response either to polyclonal or to allogeneic stimulation wasevaluated in a mixed lymphocyte reaction (MLR) assay.

Briefly, CFSE labelled CD4⁺ T cells were first stimulated with poly I:Cmatured allogeneic dendritic cells in presence of different dose ofIFNs. At 5 days post activation, the CFSE fluorescence dilution wasanalyzed.

As shown in FIG. 2, IFN-α2a inhibits the proliferation of stimulatedCD4⁺ T cells, while IFN type III exhibits no ability to suppress theirproliferation. Of note, when the MLR was performed in the presence ofanti-interferon type I receptor antibody, CD4⁺ T cells exhibit a greaterproliferation. Thus, IFN-type I but not IFN type III inhibit theproliferation of allo-activated CD4⁺ T cells.

Moreover, the analysis of mRNA levels of the interferon-induced genes(ISG), IFIT1, MX1 and OASL in IFNs treated CD4⁺ T cells confirmed thelack or minimal sensitivity of CD4⁺ T cells to interferon type III.

Indeed, as shown in FIG. 3, ISGs are induced only in CD4⁺ T cellsstimulated with IFN-α2a. Thus, IFN-α2a but not IFN-type III induce theexpression of ISGs in CD4⁺ T cells.

Because the Jak-STAT1/2 pathway being the major regulators of thetranscription of ISG, the inventors have analyzed the phosphorylationlevels of Stat1 proteins in response to IFN-type I, or interferon typeIII within CD4⁺ T cells.

As shown in FIG. 4, only IFN-α2a was able to stimulate thephosphorylation of Statl within CD4⁺ T cells. Therefore, IFN-α2a but notIFN-type III induces tyrosine phosphorylation of STAT1 in CD4⁺ T cells.

Induction of Chronic Immune Activation in Presence of Type I and IIIInterferons.

Because chronic immune activation has been reasoned to be a significantcontributor to disease progression in HIV-1-infected subjects, it ispossible to monitor disease progression by measuring the expression ofactivation markers on CD4⁺ T cell surface. Thus, the inventors haveevaluated, by flow cytometry, the capacity of both IFNs to increase theCD38 expression on stimulated CD4⁺ T cells.

As shown in FIG. 5, only IFN-α2a was able to enhance the expression ofCD38 on stimulated CD4⁺ T cells.

Collectively, these ex vivo experiments show that while exhibitinganti-viral activity, as does IFN-α, interferon type III, by contrast tothe immunosuppressive IFN-α, have no effect on CD4⁺ T cell activationand proliferation. Indeed, interferon type III do not inhibit theinitiation of the adaptative immune reaction as do IFN-α2a.

In conclusion, while interferon type I and type III are induced by thesame viral stimulating factors and exhibit similar signature profiles,their biological activity appears not redundant but rathercomplementary. Indeed, following viral infection, during the innatephase of the immune response, interferons type III exert their antiviraleffects in mucosal sites whereas IFN-α act more systemically in thewhole organism. Furthermore, the subsequent adaptive immune reaction isinhibited at its initiation level by the immunosuppressive effect of theIFN-α on activated CD4⁺ T cells.

Example 2: Ex Vivo Generation and Expansion of Antigen (Ag) SpecificCD8⁺ HLA-E Restricted T Cells Materials and Methods Human Blood Sample.

Blood samples from healthy individuals originated from EtablissementFrancais du Sang (EFS, Paris). Blood cells are collected using standardprocedures.

Cell Purification and Culture

Peripheral blood mononuclear cells (PBMCs) are isolated by densitygradient centrifugation on Ficoll-Hypaque (Pharmacia). PBMCs are usedeither as fresh cells or stored frozen in liquid nitrogen. T-cellsubsets and T cell-depleted accessory cells (ΔCD3 cells) are isolatedfrom either fresh or frozen PBMCs. T cell-depleted accessory cells (ΔCD3cells) are isolated by negative selection from PBMCs by incubation withanti-CD3-coated Dynabeads (Dynal Biotech) and are irradiated at 3000 rad(referred to as ΔCD3-feeder). Naïve CD8⁺ T cells were isolated fromPBMCs by negative selection using a MACS system. CD14⁺ monocytes areisolated from PBMCs by positive selection using a MACS system. T cellsubsets are cultured either in IMDM supplemented with 5% SVF, 100 IU/mlpenicillin/streptomycin, 1 mM sodium pyruvate, 1 mM nonessential aminoacids, glutamax and 10 mM HEPES (IMDM-5 media) in hypoxia 2%.

Freezing and Thawing of Cells

Cells were frozen in FBS containing 10% DMSO. Cryo tubes were placed inCoolCell (Biocision) freezing containers and incubated at −80° C. After2 days tubes were transferred to liquid nitrogen and stored untilrequired. Thawing of cells was performed by placing cryo tubes in a 37°C. water bath for approximately 30 seconds. Next, cell suspension wasmixed with equivalent volume of pre-warmed media and subsequentlytransferred to falcon tubes containing the same medium. Cells werepelleted by centrifugation (300 g, 3 min) to remove DMSO. The cellpellet was resuspended in cell culture medium

Dendritic Cell Generation

Monocytes were cultured in RPMI supplemented with 10% heat-inactivatedFetal Bovine Serum, 2 mM L-glutamine, 1% penicillin and streptomycinsolution (RPMI medium), in presence of IL-4 (20 ng/ml) and GM-CSF (20ng/ml). At day 6, DC were matured overnight in different cocktails: a(IL-113 (2 ng/ml) IL-6 (30 ng/ml), PGE2 (1 microg/ml) and TNF-α (10ng/ml), LPS 250 ng/ml, Poly I:C (150 ng/ml).

In Vitro Generation of TAP-Inhibited Stimulator Cells for MLR Assay

Matured DC, obtained as described above, are electroporated with 20 μgof RNA synthesized from the pGem4Z vector containing the UL49.5 genefrom BHV-1 (see, for example, Lampen et al. (2010) J Immunol.185(11):6508-17).

Induction and Expansion of Human Ag-Specific CD8 T Cells HLA-ERestricted

TAP-inhibited mature DCs (TAP-mDC) were pulsed with 50 μg/ml synthesizedpeptide. Then DCs were mixed with naive CD8 T cells at a ratio 1:10.IL-21 (30 ng/ml) was immediately added after the culture was initiated.After 3 days, half of medium were exchanged and 30 ng/ml IL-21, 20 ng/mLinterleukin 15 (IL-15) and 500 ng/mL soluble, Fc-fused IL15-Receptoralpha (sIL15Ra-Fc, R&D Systems) were added. After 10 days of coculture,T cells were restimulated with peptides pulsed TAP-inhibited mature DCsin presence of IL-21, IL-15 and Fc-fused IL15-Receptor alpha. IL-2 (50IU/ml) and IL-7 (10 ng/ml) were added 1 day after the second stimulationto further facilitate expansion of activated Ag-specific T cells.Peptide-specific expansion of T cells was monitored by flow cytometricanalysis using MHC-peptide pentamer.

Flow Cytometry Analysis

T cells were transferred per v-bottomed 96-well, washed (300 g, 2 min)and stained in 100 μL FACS buffer (PBS, 3% FBS, 2 mM EDTA) containingrespective peptide-MHC pentamers (1:10, Prolmmune) for 1 hour at 4° C.Cells were washed three times in FACS buffer and subjected to flowcytometric analysis.

Appropriate isotype control Abs are used for each staining combination.Samples are acquired on a BD LSR FORTESSA flow cytometer using BDFACSDIVA 8.0.1 software (Becton Dickinson). Results are expressed inpercentage (%) or in mean fluorescence intensity (MFI).

Results

Recent advances in the field of SIV vaccinology have highlighted therole of MHC-1b/E-restricted CD8⁺ T cell responses in controlling SIVinfection in rhesus macaques, thereby raising the possibility that theadoptive transfert of HLA-E-restricted CD8⁺ T cells could be benificialin controlling HIV-1 infection. The inventors thus established anexperimental procedure to generate and expand autologous CD8⁺ T celllines directed to peptide presented by HLA-E, using as HLA-E peptide,the CMV UL40-derived peptide (VMAPRTLIL, SEQ ID NO: 5) and as stimulatorcells, a TAP-inhibited mature DC. The use of VMAPRTLVL-HLA-E pentamer(VMAPRTLVL, SEQ ID NO: 6) allows to assess specific T cell expansion.

As shown in FIG. 6, following two rounds of stimulation, 72% CD8⁺ Tcells in culture were tetramer positive, suggesting that the inventorshave developed a culture system that facilates the expansion and thegeneration of Ag-specific CD8⁺ T cells HLA-E restricted.

Such ex vivo expanded cellular material represent per se an example ofactive principle for adoptive T cell therapy.

Example 3: Prophylactic Vaccine to SIV in Macaques with RecombinantRhCMV/SIV Vectors

The FIG. 7 is a schematic representation of the immunization protocol inmacaques.

Immunization Protocol (DNA Vaccination)

The CD8 vaccine composition is in a form adapted to intramuscularadministration and comprises RhCMV/SIV vectors (see Hansen et al. (2013)Science 24; 340(6135):1237874; Hansen et al. (2016) Science; 351(6274)).Chinese rhesus macaques receive 2 intramuscular (i.m.) injections ofsaid CD8 vaccine composition at days 0 and 14.

At days −7, −3, 3, 11, 38, 45, 52, 59, 66, 73 and 80, macaques receive(i.p.) injection of interferon lambda 1 (50-100 μg) and/or anti-IFN-αantibodies (PBL, 100 μg/kg).

Pre-Challenge

Optionally, macaques receive intra-rectal injection of non-infectiousdose of SIV or an attenuated SIV (e.g., SIV depleted in protein net).

Challenge

At days 45, 52, 59, 66, 73, and 80 macaques receive intra-rectalinjection of suboptimal dose of SlVmac 239.

Acquisition of SIV infection is determined as a plasma viral load >30copy eq/mL and/or development of an immune reaction to SIV Vif (i.e., anantigen not included in the RhCMV/SIV vector).

Example 4: Prophylactic Vaccine to SIV in Macaques with Inactivated SIVand Living Lactobacillus plantarum

The FIG. 8 is a schematic representation of the oral immunizationprotocol in macaques.

Oral Priming Immunization Protocol

The CD8 vaccine composition is in a form adapted to intragastricallyadministration and comprises: 4×10⁷ copies/mL of an inactivated SIV and3×10⁹ cfu/mL of living Lactobacillus plantarum in maltodextrin (20%)solution. Chinese rhesus macaques receive 30 mL of said CD8 vaccinecomposition and then 25 mL of the same composition every 30 min for 3hours, at days 0, 1, 3, 5, 7 and 28, 29.

At day −3 and before each series of immunizations at days 0, 28 and 29,macaques receive (i.p.) injections of a combination of differentreagents according to the protocol as described hereinafter:

-   -   Protocol 1: nothing    -   Protocol 2: poly I:C (100 μg/kg) and interferon lambda 1 (50-100        μg)    -   Protocol 3: polyclonal anti-IFN-α antibodies (PBL, 100 μg/kg)        and poly I:C (100 μg/kg)    -   Protocol 4: polyclonal anti-IFN-α antibodies (PBL, 100 μg/kg),        poly I:C (100 μg/kg) and interferon lambda 1 (50-100 μg)

At day 57, macaques receive injection of polyclonal anti-IFN-αantibodies (PBL, 100 μg/kg), poly I:C (100 μg/kg) and/or interferonlambda 1 (50-100 μg).

Bosting Immunization (Pre-Challenge)

At day 60, macaques receive intra-rectal injection of non-infectiousdose of SIV or an attenuated SIV (e.g., SIV depleted in protein net).

Immune Response Analysis

Anti-SIV immune response is analyzed at day 25, 57 and 80. The analysisof the anti-SIV immune response comprises the monitoring of:

-   -   Plasma SIV IgM/IgG/IgA antibody titers, and/or    -   SIV Gag specific CD8⁺ T cell and CD8⁺ T cell-mediated anti-viral        activity.

Challenge

At day 90, macaques receive intra-rectal injection of an infectious doseof SIV only if an anti-SIV specific CD8⁺ suppressive T cells (asdescribed in the present invention) is observed.

After challenge, anti-SIV immune responses and plasma viremia aremonitored every two weeks.

Example 5: Prophylactic Vaccine to HIV in BLT Mice

The FIG. 9 is a schematic representation of the oral immunizationprotocol in BLT mice.

BLT mice are valuable humanized models for the study of HIV infection.Indeed, BLT mice recapitulate important aspects of human immunity,including T cell immunity (Marshall E. Karpel et al., Curr Opin Virol.2015 August; 13: 75-80).

Oral Immunization Protocol

The CD8 vaccine composition is in a form adapted to intragastricallyadministration and comprises: 4×10⁷ copies/mL of an inactivated HIV-1and 3×10⁹ cfu/mL of living Lactobacillus plantarum in maltodextrin (20%)solution. BLT mice receive 0.2 mL of said CD8 vaccine composition andthen 0.2 mL of the same composition every 30 min for 3 hours, at days 0,1, 3, 5, 7 and 28, 29.

At day −3 and before each series of immunizations at days 0, 28 and 29,BLT mice receive (i.p.) injections of a combination of differentreagents according to the protocol as described hereinafter:

-   -   Protocol 1: nothing    -   Protocol 2: poly I:C (100 μg/kg) and interferon lambda 1 (50-100        μg)    -   Protocol 3: polyclonal anti-IFN-α antibodies (PBL, 100 μg/kg)        and poly I:C (100 μg/kg)    -   Protocol 4: polyclonal anti-IFN-α antibodies (PBL, 100 μg/kg),        poly I:C (100 μg/kg) and interferon lambda 1 (50-100 μg)

At day 57, BLT mice receive injection of polyclonal anti-IFN-αantibodies (PBL, 100 μg/kg), poly I:C (100 μg/kg) and/or interferonlambda 1 (50-100 μg).

Bosting Immunization (Pre-Challenge)

At day 60, BLT mice receive intra-rectal injection of non-infectiousdose of HIV-1 or an attenuated HIV-1 (e.g., HIV-1 depleted in proteinnet).

Immune Response Analysis

Anti-HIV immune response is analyzed at day 25, 57 and 80. The analysisof the anti-SIV immune response comprises the monitoring of:

-   -   Plasma HIV IgM/IgG/IgA antibody titers, and/or    -   HIV Gag specific CD8⁺ T cell and CD8⁺ T cell-mediated anti-viral        activity.

Challenge

At day 90, BLT mice receive intra-rectal injection of an infectious doseof HIV-1 only if an anti-HIV-1 specific CD8⁺ suppressive T cells (asdescribed in the present invention) is observed.

After challenge, anti-HIV-1 immune responses and plasma viremia aremonitored every two weeks.

Example 6: Ex Vivo Generation and Expansion of Antigen (Ag) SpecificCD8+ HLA-E Restricted T Cells Using HLA-E*01033-Transfected Derivativeof K562 Cell Line Materials and Methods Human Blood Sample

Blood samples from healthy individuals originated from EtablissementFrancais du Sang (EFS, Paris). Blood cells were collected using standardprocedures.

Cell Purification and Culture

Peripheral blood mononuclear cells (PBMCs) were isolated by densitygradient centrifugation on Ficoll-Hypaque (Pharmacia). PBMCs were usedeither as fresh cells or stored frozen in liquid nitrogen. Naïve CD8⁺ Tcells were isolated from PBMCs by negative selection using a MACSsystem. T cell subsets were cultured either in IMDM supplemented with 5%SVF, 100 IU/ml penicillin/streptomycin, 1 mM sodium pyruvate, 1 mMnonessential amino acids, glutamax and 10 mM HEPES (IMDM-5 media) inhypoxia 2%.

HLA-E*01033-transfected derivative of K562 (K562/HLA-E) cell line weremaintained in RPMI 1640 medium (Lonza, Basel, Switzerland) supplementedwith 10% fetal bovine serum, 2 mM L-glutamine, 25 mM HEPES andantibiotics in presence of 0.4 mg/ml G-418 (Calbiochem, San Diego,Calif.). When used as antigen-presenting cells, pulsed K562/HLA-E cellline was irradiated at 80000 rad.

Peptide-HLA Molecule Binding Assays

Peptide binding was assessed by HLA-E stabilization assays usingHLA-E*01033-transfected derivative of K562 (K562/HLA-E) cell lines.Briefly, cells were re-suspended in serum-free medium at 1×10⁶ cells/ml.Where appropriate, a peptide (see Table 1) was added. After overnightincubation at 26° C., cells were washed with PBS to remove freepeptides. Next, HLA surface expression was monitored after staining withanti-HLA-E mAb. Analysis was done on a FACScalibur cytometer (BDBiosciences). Results are presented as the MFI of cells stained withanti-HLA-E mAb.

Generation and Expansion of Peptide-Specific HLA-E Restricted CD8⁺ TCell Lines

Naïve CD8⁺ T were cultured in the presence of irradiated K562/HLA-E cellline as antigen-presenting cells (at 0.5×10⁶/ml) pulsed with theappropriate peptide (10 μM) in complete medium supplemented with IL-21(30 ng/ml). After 3 days, half of medium was exchanged and 30 ng/mlIL-21, 20 ng/mL interleukin 15 (IL-15) and 500 ng/mL soluble, Fc-fusedIL15-Receptor alpha (sIL15Ra-Fc, R&D Systems) were added. Restimulationwas done after 10 days with irradiated K562/HLA-E cells pulsed with thecorresponding peptide in presence of IL-21, IL-15 and Fc-fusedIL15-Receptor alpha. IL-2 (50 IU/ml) and IL-7 (10 ng/ml) were added 1day after the second stimulation to further facilitate expansion ofactivated Ag-specific T cells. Then, cultures were stimulated weekly for4-10 weeks. Peptide-specific expansion of T cells was monitored by flowcytometric analysis using a cytotoxic assay.

Cytotoxicity Assay

A cell-based Flow Cytometry assay was used to measured specificcytotoxic activity of the peptide-specific HLA-E restricted CD8⁺ T celllines ex vivo generated. Briefly, CFSE labelled targets were incubatedovernight at 26° C. (K562/HLA-E) in presence or absence of syntheticpeptides and co-cultured with the CD8⁺ T cell lines for 5 hours atdifferent ratios (10:1 and 1:1). Control tubes (target cells withoutCD8⁺ T cell lines) were also assayed to determine the spontaneous celldeath. After 5 hours of co-culture, cells were stained with 7-AAD. Fordata analysis, the CFSE-positive target cells were examined for celldeath by uptake of 7-AAD. CFSE and 7-AAD double positive cells wereconsidered to be dead target cells. The percentage of specific cytotoxicactivity was subsequently calculated using the following equation:

Cytotoxicity (%)=Target cell death−Spontaneous death×100−Spontaneousdeath×100

Results

HLA-E Expression on K562 Cell Line after Peptide Loading

Untransfected K562 cells do not display surface expression of HLA-E asassessed by flow cytometry as well as HLA-E transfected K562 in theabsence of peptide loading (see Table 1). After pulsing K562/HLA-Eovernight at 26° C. with specific HLA-E peptide from different origin,HLA-E surface expression was induced (see Table 1).

TABLE 1 Peptide-HLA molecule binding assay Cells Peptides MFIUntransfected K562 Without peptide 1250 HLA-E Transfected K562Without peptide 1400 HLA-E Transfected K562 HCMV derived peptide 6250VMAPRTLIL (SEQ ID NO: 5) HLA-E Transfected K562 EBV derived peptide 5860SQAPLPCVL (SEQ ID NO: 14) HLA-E Transfected K562 MBt derived peptide7523 VMATRRNVL (SEQ ID NO: 15) HLA-E Transfected K562HIV-1 Pol derived peptide 7320 PEIVIYDYM (SEQ ID NO: 16)HLA-E Transfected K562 HIV-1 Pol derived peptide 6580RIRTWKSLV (SEQ ID NO: 17) HLA-E Transfected K562HIV-1 Gag derived peptide 8240 RMYSPVSIL (SEQ ID NO: 1)HLA-E Transfected K562 HIV-1 Gag derived peptide 8530TALSEGATP (SEQ ID NO: 3)

Detection of Antigen Specific HLA-E Restricted CD8⁺ T Cells UsingCytotoxicity Assay

Seeing cell proliferation in the long-term stimulated cultures, theinventors wanted to estimate their specific functional activity usingcytotoxicity assay. K562/HLA-E peptide pulsed cells induced theactivation and the expansion of antigen-specific CD8⁺ T cell lines,since cells that have proliferated exhibit a high cytotoxic responseagainst the candidate antigens, while no significant activity against anirrelevant antigen (see Table 2).

TABLE 2 specific cytotoxic activity of the expanded CD8+ T cellsPeptide specific HLA-E % cytotoxicity against % cytotoxicity against anrestricted CD8+ the relevant peptide irrelevant peptide T cell linesRatio 10:1 Ratio 1:1 Ratio 10:1 Ratio 1:1 HCMV derived peptide 75 32 12 8 VMAPRTLIL EBV derived peptide 82 35 15 10 SQAPLPCVLMBt derived peptide 67 28  9  5 VMATRRNVL HIV-1 Pol derived peptide 8735 17  8 PEIVIYDYM HIV-1 Pol derived peptide 73 28 16 10 RIRTWKSLVHIV-1 Gag derived peptide 82 33 15  9 RMYSPVSILHIV-1 Gag derived peptide 86 32 17 10 TALSEGATP

Example 7: Critical Pathogenic Role of IFN-α in Human HIV-1 InfectionMaterial and Methods

Cryopreserved PBMCs were thawed in RPMI 1640 with 10% fetal bovine serum(FBS) and washed in FACS buffer. Phenotypic staining was performed on10⁶ cells by incubation with a viability marker (AmCyan live-dead kitfrom Invitrogen) and with antibodies conjugated to CD3, CD4, CD8,CD45RA, CCR7. Subsequently, cells were washed, fixed with 4%paraformaldehyde for 5 min, washed, and acquired with an AURORAcytometer (Cytek).

Frozen serums were thawed at 4° C. and centrifuged at 4000 G for 10 minat 4° C. IFN-α serum concentrations were measured using the highsensitivity Simoa® technology (Digital ELISA technology) (Quanterix).

Results Comparison of Central Memory (CM) CD8⁺ T Cell Distributions inHIV-1-Infected Subjects

In study of chronically HIV-1-infected subjects, the following groupswere studied:

(i) elite controllers (EC) who naturally suppress HIV-1 in the absenceof combined antiretroviral therapy treatment (c-ART)(ii) non-controllers before (pre-cART) and after cART (post-cART)treatment, and(iii) a cohort of age-matched healthy donor (HD) subjects.

The relative frequencies of the CM populations within the CD8⁺ T cellcompartments were evaluated in each of the subject groups.

The gating strategy to define this subset is the following. Briefly,singlet cells were defined, followed by gating on lymphocytes and livecells. Among the live cells, CD3⁺ T lymphocytes were identified,followed by the definition of CD8+ subpopulations. Subsequently, theexpression of CD45RA and CCR7 was analyzed in the CD8⁺ T lymphocytes.Central memory T cells (TCM) are CD45RA-CCR7+.

FIG. 10A shows that the level of CM CD8⁺ cells was significantly lowerin non-controllers before cART than in other groups. Moreover, combinedantiretroviral therapy (cART) results in increase of CM CD8⁺ cells.

Comparison of Serum IFN-α Levels in HIV-1-Infected Subjects

Serum levels of IFN-α was measured in the 4 groups. FIG. 10B shows thatthe non-controller patients have significantly increased serum IFN-αlevels before treatment compared with after treatment.

IFN-α Inversely Correlates with the Percentage of CM CD8⁺ Cells inHIV-Infected Patients without Treatment

In the population of HIV infected patients (EC pre-cART patients), theinventors explored the potential correlations between the level of CMCD8⁺ cells and the serum IFN-α levels. In this study, there was asignificant negative correlation between the frequency of CM CD8⁺ cellsand serum IFN-α levels (spearman correlation r=−0.667; p<0.005). Thisreflects the critical pathogenic effect of IFN-α on T cell proliferationin secondary organs (see FIG. 10C).

Example 8: Prophylactic Vaccine to SIV in Macaques with Inactivated SIVand Living Lactobacillus plantarum

This protocol comprises two steps described below.

FIG. 11 is a schematic representation of the protocol scheme of step 1.

Step 1: Identification of the Most Efficient Regimen for Induction ofVirus Specific CD8+ Suppressive T Cells in Rhesus Macaques (RM)

Immunization protocol is based on the experimental work performed onChinese rhesus macaques and described in Lu et al. (2012) Cell Rep.2(6), 1736-46. This protocol is comprised of three steps:

1) oral priming with preparation containing inactivated SIVmac239, asactivate principle, and living Lactobacillus plantarum (LP), asadjuvant,2) oral boosting with the same preparation and3) intrarectal boosting with non-infectious doses of living virus.

Eight male RM, 2 RM of Chinese origin and 6 of Indian origin areincluded.

Oral priming immunization is carried out on 5 consecutive days. Eachday, monkeys are intragastrically administered 30 ml of a preparationcontaining 4×10⁷ copies/ml of inactivated SIV and 3×10⁹ cfu/ml of livingLP in maltodextrin (20%) solution. Then they receive 25 ml of the samepreparation intragastrically every 30 min for 3 hours.

Oral boosting immunization is administered at day 28 and day 29 and ifnecessary at day 60 and day 61.

Intrarectal boost is performed twice by a 2-week interval from day 90.Monkeys of Indian origin additionally receive an intraperitoneal (IP)injection of poly I:C and lambda IFN at day −2, day 3 during the oralpriming and two days before each oral boosting, i.e. at day 26 and ifnecessary at day 58 and one day before IR boosts.

The 6 RM of Indian origin are distributed in two groups (B and C)depending on when the anti-IFNα antibody is added during theimmunization. For group B anti-IFNα antibody is administered at day 32,and if necessary at day 64, one day before each IR boosts. For group C,anti-IFNα antibody is administered only five days after the first IRboost. For all macaques, induction of virus specific CD8+ suppressive Tcells is monitored at day 50 and if necessary at day 80 and at day 126.Plasma viral loads are followed weekly after the IR boosts.

Step 2: Evaluation of the Vaccine Efficacy by Rectal Challenge withSIVmac239.

Only monkeys who mount virus-specific CD8+ suppressive T cells arechallenged intrarectally with high infectious dose of SIVmac239 (100,000TCID50). Plasma viral loads are followed every week for 6 weeks.

Example 9: Prophylactic Vaccine to HIV in BSLT Mice

This protocol comprises two steps described below.

FIG. 12 is a schematic representation of the protocol scheme of step 1.

Step 1: Identification of the Most Efficient Regimen for Induction ofAnti-HIV-1 Specific CD8+ Suppressive T Cells in BSLT Mice

Immunization protocol is based on the experimental work performed onChinese rhesus macaques and described in Lu et al. (2012) Cell Rep.2(6), 1736-46. This protocol is comprised of three steps:

1) oral priming via intragastric route with preparation containinginactivated HIV-1, as activate principle, and living Lactobacillusplantarum (LP), as adjuvant;2) oral boosting via intragastric route with the same preparation;3) intrarectal boosting with low doses of living virus.

Four groups of 10 mice (A, B, C and D) are included. Group D is acontrol group to monitor the effectiveness of the challenge. Mice ofthis group D receive no immunization or is immunized with PBS.

Oral priming immunization via intragastric route consists of daily oralintake of inactivated HIV-1 and living LP in maltodextrin (20%) solutionover a period of 5 days. Each day, mice receive the same preparationintragastrically, 3 times every 30 minutes over 1 hour. Mice receive 200mcl of the preparation.

Oral boosting immunization via intragastric route is administered at day28 and day 29 and, if necessary, at day 60 and day 61.

Intrarectal boost is performed twice by a 2-week interval at day 90 andday 104. Group B and C additionally receive an intraperitoneal injectionof poly I:C and lambda IFN (100-500 mcl) at day −2, day 3 during theoral priming via intragastric route and two days before each oralboosting via intragastric route, i.e. at day 26 and, if necessary, atday 58 and one day before the IR boosts (day 89 and day 103).

Group B and C differ depending on when the anti-IFNα antibody is addedduring the immunization. For group B anti-IFNα antibody is administeredat day 32 during the oral boost via intragastric route and, ifnecessary, at day 64 during the oral boost via intragastric route. Forgroup C anti-IFNα antibody is administered only five days after the IRboosts. For all mice, induction of virus-specific CD8⁺ suppressive Tcells is monitored at day 50 and, if necessary, at day 80 and at day126. Plasma viral loads are followed weekly after the IR boosts.

Step 2: Evaluation of the Vaccine Effectiveness by Rectal Challenge withHIV-1

Two months after the last antibody anti-IFNα administration, only micewhich mount virus-specific CD8+ suppressive T cells are challengedintrarectally with high infectious dose of HIV-1 (100,000 TCID50).Plasma viral loads are followed every week for 6 weeks.

1. A method for preventing acquired immune deficiency syndrome (AIDS) ina subject in need thereof, comprising administering to the subject: 1) aCD8 vaccine specific for at least one human immunodeficiency virus (HIV)antigen, 2) optionally interferon-alpha blocking agent, and 3) a typeIII interferon and/or an agent stimulating the production of type IIIinterferon.
 2. The method for preventing according to claim 1, whereinthe type III interferon comprises at least one IFN-λ selected from thegroup of IFN-λ1, IFN-λ2 IFN-λ3 and IFN-λ4, and wherein the agentstimulating the production of type III interferon comprises at least oneTLR ligand, RIG-I ligand, and/or MDA5 ligand.
 3. The method forpreventing according to claim 2, wherein the interferon-alpha blockingagent is selected from the group of: an agent neutralizing circulatingalpha interferon, an agent blocking interferon-alpha signaling, an agentdepleting IFN-α producing cells, and/or an agent blocking IFN-αproduction, wherein the agent neutralizing circulating alpha interferonis selected from the group comprising active anti-IFN-α vaccineincluding antiferon or passive anti-IFN-α vaccine including anti-IFN-αantibodies or anti-IFN-α hyper-immune serum, wherein the blocking agentof interferon-alpha signaling is selected from the group of anti-type Iinterferon R1 or R2 antibodies or from interferon-alpha endogenousregulators including SOSC1 or aryl hydrocarbon receptors, wherein theagent depleting IFN-α producing cells is an agent depleting plasmacytoiddendritic cells (pDCs), and wherein the agent blocking IFN-α productionis an agent blocking the production of IFN-α by pDCs.
 4. The method forpreventing according to claim 1, wherein the interferon-alpha blockingagent is selected from the group of: an agent neutralizing circulatingalpha interferon, an agent blocking interferon-alpha signaling, an agentdepleting IFN-α producing cells, and/or an agent blocking IFN-αproduction, wherein the agent neutralizing circulating alpha interferonis selected from the group comprising active anti-IFN-α vaccineincluding antiferon or passive anti-IFN-α vaccine including anti-IFN-αantibodies or anti-IFN-α hyper-immune serum, wherein the blocking agentof interferon-alpha signaling is selected from the group of anti-type Iinterferon R1 or R2 antibodies or from interferon-alpha endogenousregulators including SOSC1 or aryl hydrocarbon receptors, wherein theagent depleting IFN-α producing cells is an agent depleting plasmacytoiddendritic cells (pDCs), and wherein the agent blocking IFN-α productionis an agent blocking the production of IFN-α by pDCs.
 5. The method forpreventing according to claim 1, wherein the CD8 vaccine elicits orcomprises suppressor MHC-1b/E-restricted CD8⁺ T cells
 6. The method forpreventing according to claim 1, wherein the CD8 vaccine elicits orcomprises suppressor MHC-1b/E-restricted CD8⁺ T cells, and wherein thesuppressor MHC-1b/E-restricted CD8⁺ T cells are generated by ex vivo orin vivo induction of HLA-1a-deprived dendritic cells.
 7. The method forpreventing according to claim 1, wherein the CD8 vaccine elicits orcomprises suppressor MHC-1b/E-restricted CD8⁺ T cells, wherein thesuppressor MHC-1b/E-restricted CD8⁺ T cells are generated by ex vivo orin vivo induction of HLA-1a-deprived dendritic cells, and wherein theHLA-1a-deprived dendritic cells are obtained by an agent inhibiting TAPexpression or activity.
 8. The method for preventing according to claim1, wherein the CD8 vaccine is an active vaccine, wherein the CD8 vaccineis a live viral vector comprising at least one HIV antigen, and whereinthe live viral vector is selected from the group of cytomegalovirus,lentivirus, vaccinia virus, adenovirus or plasmid.
 9. The method forpreventing according to claim 1, wherein the CD8 vaccine is an activevaccine, and wherein the CD8 vaccine is a cytomegalovirus (CMV) vectorcomprising: a first nucleic acid sequence encoding at least one HIVantigen, optionally a second nucleic acid sequence comprising a firstmicroRNA recognition element (MRE) operably linked to a CMV gene that isessential or augmenting for CMV growth, wherein the MRE silencesexpression in the presence of a microRNA that is expressed by a cell ofendothelial lineage; and wherein the CMV vector does not express anactive UL128 protein or ortholog thereof; does not express an activeUL130 protein or ortholog thereof; does not express an active UL146 orortholog thereof; does not express an active UL147 protein or orthologthereof, and wherein the CMV vector expresses at least one active UL40protein or an ortholog thereof; expresses at least one active US27protein or an ortholog thereof and/or expresses at least one active US28protein or an ortholog thereof.
 10. The method for preventing accordingto claim 1, wherein the CD8 vaccine is a cytomegalovirus (CMV) vector,and wherein the CMV vector is a human CMV (hCMV).
 11. The method forpreventing according to claim 1, wherein the CD8 vaccine is an activevaccine, and wherein the CD8 vaccine comprises at least one HIV antigenand a non-pathogenic bacterium.
 12. The method for preventing accordingto claim 1, wherein the CD8 vaccine comprises at least one HIV antigenand a non-pathogenic bacterium, wherein the HIV antigen is selected fromthe group of virus, virus particles, virus-like particles, recombinantvirus, recombinant virus particles, conjugate viral proteins andconcatemer viral proteins, and wherein said virus, virus particles orsaid recombinant virus particles are attenuated or inactivated.
 13. Themethod for preventing according to claim 1, wherein the CD8 vaccinecomprises at least one HIV antigen and a non-pathogenic bacterium,wherein the non-pathogenic bacterium is living, and wherein saidnon-pathogenic bacterium is selected from attenuated or inactivatedpathogenic bacteria.
 14. The method for preventing according to claim 1,wherein the CD8 vaccine comprises at least one HIV antigen and anon-pathogenic bacterium, and wherein the non-pathogenic bacterium is aLactobacillus bacterium.
 15. The method for preventing according toclaim 1, wherein the CD8 vaccine comprises at least one HIV antigen anda non-pathogenic bacterium, and wherein the non-pathogenic bacterium isLactobacillus plantarum.
 16. The method for preventing according claim1, wherein the CD8 vaccine is an active vaccine, and wherein the CD8vaccine is an ex vivo generated dendritic, natural killer or B cellpopulation presenting MHC-1b/E-restricted and MHC-II restrictedantigens, and wherein the MHC-1b/E-restricted antigen is an HIV antigen.17. The method for preventing according to claim 1, wherein the CD8vaccine is a passive vaccine, and wherein the CD8 vaccine is an ex vivogenerated autologous MHC-1b/E-restricted CD8⁺ T cell population, andwherein the MHC-1b/E-restricted CD8⁺ T cell population recognizes anMHC-1b/E-restricted HIV antigen.
 18. The method for preventing accordingto claim 1, wherein the HIV antigen is derived from any HIV strain, andwherein the HIV antigen is selected from the group consisting of HIVgag, HIV env, HIV rev, HIV tat, HIV nef, HIV pol, and HIV vif antigens.19. The method for preventing according claim 1, wherein the HIV antigenis an HIV-derived HLA-E-binding antigen.
 20. The method for preventingaccording to claim 1, wherein the HIV antigen is a HIV-derivedHLA-E-binding antigen, and wherein the HIV-derived HLA-E-binding antigenis selected from the antigens of SEQ ID NO: 1 to SEQ ID NO: 4.